Animal Model Having a Chimeric Human Liver and Susceptible to Human Hepatitis C Virus Infection

ABSTRACT

The present invention features a non-human animal model that is susceptible to infection by human hepatotrophic pathogens, particularly human hepatitis C virus (HCV). The model is based on a non-human, immunocompromised transgenic animal having a human-mouse chimeric liver, where the transgene provides for expression of a urokinase-type plasminogen activator in the liver. The invention also features methods for identifying candidate therapeutic agents, e.g., agents having antiviral activity against HCV infection. The animals of the invention are also useful in assessing toxicity of various agents, as well as the activity of agents in decreasing blood lipids.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of PCT application serial no.PCT/CA01/00350, filed Mar. 16, 2001, and a continuation-in-part of U.S.application Ser. No. 09/528,120, filed Mar. 17, 2000, each of whichapplications are incorporated herein by reference in entirety.

FIELD OF THE INVENTION

The present invention relates generally to animals useful as a model ofinfection by a viral pathogen, such as hepatitis virus, as well as inassessment of toxicity and evaluation of therapies for hyperlipidemia.

BACKGROUND OF THE INVENTION

Human liver disease caused by the hepatitis C virus (HCV) has emergedover the past decade as one of the most difficult challenges facing theworldwide medical community. Elucidation of the viral sequence in 1989(Choo, et al. Science 244, 359-361 (1989)) initiated the era ofconcerted study of HCV; presently it is estimated that up to 175,000,000people are infected (Sarbah, et al. Cell 62, 447-456 (1990)). HCV is themost common type of chronic viral hepatitis with an estimated prevalenceof 1-2% in developed countries. Chronic HCV hepatitis leads to livercirrhosis in at least 25% of affected patients and after development ofcirrhosis it is estimated that hepatocellular carcinoma develops in 1-4%of patients each year. In North America HCV is currently the most commonindication for liver transplantation.

Currently antiviral therapy with combination interferon and ribavirin iseffective in selected patients, but many either fail to respond ortolerate therapy poorly, underscoring a need for improvement. Sustainedresponse rates for interferon monotherapy range from 20-25%, whilecombination therapy with interferon and ribavirin has shown sustainedresponse rates of up to 40%. Although newer antiviral drugs targetingdifferent parts of the viral genome are under development, progress hasbeen severely hampered by the lack of a robust cost-effective animalmodel of HCV. The only natural hosts for HCV are humans and chimpanzees,neither of which is suitable for large scale antiviral testing.

The lack of a reproducible small animal model for HCV infection hasfurther limited the investigation of various immune factors contributingto the disease, as well as vaccine candidates for the immunotherapy ofchronic HCV infections. In the case of HCV infection, a number ofreports have demonstrated the presence of Th1->Th2 switch and HCVantigens specific CD4+ and CD8+ T cells in in vitro studies on T cellsisolated from the HCV infected individuals. On the other hand,non-viremic HCV infected patients have been found to stimulate strongTh1 response to multiple HCV antigens even many years after infections,suggesting that control of HCV replication may depend on effective Th1activation (Cramp et al. Gut 44:424-429 (1999)). Resolution of thesequestions to provide a better understanding of the immune response toHCV, and thus insight as to the development of effective vaccines andtherapies, can not be easily reached without a suitable animal model.

Over the past several years, significant advances have been made in thedevelopment of animal models for hepatitis B virus. However, despitetheir similar sounding names, human hepatitis B virus (HBV) and humanhepatitis C virus (HCV) are completely different viruses, and thusresearch regarding HBV infection can not be readily extrapolated to HCVinfection. Both viruses are referred to as “hepatitis” viruses primarilybecause HBV and HCV infect and replicate in the liver. Aside from this,HBV and HCV are no more alike than are HIV and EBV, which each affectthe immune system. In fact, HBV and HCV are so different that they arenot even member of the same phylogenetic family. HBV is a member of thehepadnavirus family with a genome of double-stranded DNA, whereas HCV isa member of the flavivirus family, which is based on a singlepositive-stranded RNA genome.

HBV and HCV also differ in their infectivity. HCV is less infectiousthan an equivalent dose of HBV, as evidenced by the differences inacquisition rates in hospital personnel after needlestick injuries. HBVinfections occur in 2-40% of HBV-contaminated needlestick events, whileHCV infections occur in only 3-10% of HCV-contaminated needlestickevents. These observations suggest that HCV is about three to four timesless infectious than HBV (Shapiro Surgical Clin North Amer.75(6):1047-56 (1995)).

HBV and HCV differ greatly in their requirements for replication as wellas in the viral load during infection. HBV is capable of replicating inless differentiated systems (e.g., HepG2 cells, Sells et al. Proc. Natl.Acad. Sci. USA 84:1005 (1987)). In contrast, HCV replication may dependupon the presence of nontransformed hepatocytes (see, e.g., Ito et al.J. Gen. Virol. 77:1043 (1995)). The viral titers of patients infectedwith HCV are generally lower than those of HBV-infected patients.Patients infected with HBV have levels ranging from 10⁵ to 10⁹ particlesper mL, compared to 102 to 107 particles per mL in HCV infections. Thesedifferences in viral titer may be due at least in part to the relativeclearance rates of viral particles. In addition, the number of viralcopies per cell is also very low in HCV infection (e.g., generally lessthan 20 copies per cell (Dhillon et al. Histopathology 26:297-309(1995)). This combination of low viral titers and low number of viralcopies per cell means that a significant number of human hepatocytesmust be infected and producing virus for the infection to even bedetected within serum.

The limited host range of human HBV and human HCV has proved problematicin the development of in vitro and in vivo models of infection. Humansand chimpanzees are the only animals susceptible to human HBV infection;human, chimpanzees, and tree shrews are susceptible for infection withhuman HCV (Xie et al. Virology 244:513-20 (1998), reporting transientinfection of tree shrews with HCV). Human HBV will infect isolated humanliver cells in culture (see, e.g., Sureau Arch. Virol. 8:3-14 (1993);Lampertico et al. Hepatology 13; 422-6 (1991)). HCV has been reported toinfect primary cultures of human hepatocytes; however, the cells do notsupport the production of progeny virions (Fournier et al. J Gen Virol79(Pt 10):2367-74 (1998)). The development of a satisfactory in vivomodel is required in order to provide a more clinically relevant meansfor assaying candidate therapeutic agents.

The extremely narrow host range of HBV and HCV has made it verydifficult to develop animal models. Current animal models of HBV and HCVeither do not involve the normal course of infection, require the use ofpreviously infected human liver cells, or both (see, e.g., U.S. Pat.Nos. 5,709,843; 5,652,373; 5,804,160; 5,849,288; 5,858,328; and5,866,757; describing a chimeric mouse model for HBV infection bytransplanting HBV-infected human liver cells under the mouse kidneycapsule; WO 99/16307 and Galun et al. J. Infect. Dis. 172:25-30 (1995),describing transplantation of HCV-infected human hepatocytes into liverof immunodeficient mice; Bronowicki et al. Hepatology 28:211-8 (1998),describing intraperitoneal injection of HCV-infected hematopoietic cellsinto SCID mice; and Lerta et al. Hepatology 28(4Pt2):498A (1998),describing mice transgenic for the HCV genome). Infection by human HBVis fairly well mimicked by infection of woodchucks with woodchuckhepatitis virus (WHV) and by infection of Peking ducks with duckhepatitis virus (DHV). WHV-infected woodchucks and DHV-infected duckshave been successfully used to identify drugs effective against humanHBV infection of humans. However, no analogous animal model of infectionhas been identified for human HCV.

In the absence of a practical non-human host, the most desirable animalmodel would be a chimeric animal model that allowed for infection ofhuman liver cells through the normal route of infection, preferably amouse model susceptible to viral infection through intravenousinoculation and that could support chronic infection. Unfortunately, thedevelopment of mice having chimeric livers with human hepatocytessusceptible to HBV or HCV infection, and sustaining viral replicationand virion production at clinically relevant, sustainable levels hasproven no simple matter. The field of xenogeneic liver transplantationhas moved very slowly and met with many obstacles.

In order to study neonatal bleeding disorders and hypofibrinogenemia, amouse transgenic for an albumin-urokinase-type plasminogen activatorconstruct (Alb-uPA) was developed (Heckel et al. Cell 62:447-56 (1990);Sandgren et al. Cell 66:245-56 (1991)). The Alb-uPA transgene includes amurine urokinase gene under the control of the albumin promoter,resulting in the targeting of urokinase production to the liver andproducing a profoundly hypofibrinogenemic state. This transgene was alsofound to be associated with accelerated hepatocyte death. Later workwith this transgenic animal demonstrated that individual hepatocytesthat spontaneously deleted the transgene acquired a significant survivaland replicative advantage, resulting in repopulation of the liver withthese nontransgenic cells Sandgren et al., (1991), supra). The Alb-uPAtransgenic mouse has proved amenable to transplantation with liver cellsfrom non-transgenic mice (Rhim et al. Science 263:1149-52 (1994)). TheAlb-uPA transgenic mouse was also successfully used to produce micehaving chimeric livers with rat hepatocytes (Rhim et al. Proc. Natl.Acad. Sci. USA 92:4942-6 (1995)) or woodchuck hepatocytes (Petersen etal. Proc. Natl. Acad. Sci. USA 95:310-5 (1998). However, thesedevelopments were still a long step away from the development of ananimal model susceptible to HCV infection. Production of mouse having axenogeneic transplant from another member of the Rodentia family is notnearly as difficult or unexpected as production of a mouse having axenogeneic transplant from an animal of a different family, e.g., ahuman, much less would one expect that a high degree of chimerism couldbe accomplished, or that such chimeric animals might support HCVinfection. For example, hepatocyte growth factor (HGF) is the mostpotent stimulus of hepatocyte regeneration in vivo; in comparingsequence data, mouse HGF was shown to have 98.5% amino acid sequencehomology with rat HGF, and only 90.9% with human HGF (Liu et al. Biochimet Biophys Acta 1216; 299-303 (1993)). There were no guarantees ofsuccess.

There is a need in the field for a human-mouse liver chimera susceptibleto chronic infection with HCV and with viral production at clinicallyrelevant levels. The present invention addresses this problem.

SUMMARY OF THE INVENTION

The present invention features a non-human animal model that issusceptible to infection by human hepatotrophic pathogens, particularlyhuman hepatitis C virus (HCV). The model is based on a non-human,immunocompromised transgenic animal having a human-mouse chimeric liver,where the transgene provides for expression of a urokinase-typeplasminogen activator in the liver. The invention also features methodsfor identifying candidate therapeutic agents, e.g., agents havingantiviral activity against HCV infection. The animals of the inventionare also useful in assessing toxicity of various agents, as well as theactivity of agents in decreasing blood lipids.

In one aspect the invention provides a non-human animal model that issusceptible to infection by human HCV via the normal route of infection.

In another aspect the invention provides a non-human animal model isuseful in assessing toxicity of an agent.

In another aspect the invention provides a non-human animal model isuseful in identifying agents that decrease blood lipids.

An advantage of the invention is that the animal model provides thefirst instance of an animal that is susceptible to infection by HCV viathe normal route of infection, and further that can become chronically,consistently, and stably infected at viral titers that can be equated toviral titers in HCV-infected humans.

Still another advantage of the invention is that production of theanimal model does not require obtaining or handling HCV-infected cells.Thus the invention avoids the need to obtain hepatocytes fromHCV-infected human donors or to culture and infect human hepatocytes invitro.

Another advantage of the invention is that provides for methods forassessing toxicity and drug efficacy in an in vivo setting, rather thanliver cells in vitro.

These and other objects, advantages, and features of the invention willbecome apparent to those persons skilled in the art upon reading thedetails of the animal model and methods of its use as more fullydescribed below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a Western blot of human albumin (HA) production in recipientserum samples over time in animals carrying or not carrying the Alb-uPAtransgene FIGS. 2A-2F are photographs of histochemical analysis of humanchimerism in mouse livers. FIG. 2A is a low power H&E section of controlmouse liver taken from a nontransplanted homozygous Alb-uPA liver,showing uniform cellular architecture. FIG. 2B is a low power H&Esection from a transplanted homozygous mouse, showing a large nodule oftissue compressing surrounding host-derived liver parenchyma. FIGS. 2Cand 2D show control sections of mouse and human liver respectively, bothimmunostained with an anti-human hepatocyte antibody, demonstrating theimmunohistochemical procedure clearly stains human cells, but not murinecells. FIG. 2E is a high power H&E stained section of transplantedhomozygote liver, showing a nodule of healthy hepatocytes compressingsurrounding tissue. FIG. 2F is a consecutive section immunostained forhuman hepatocyte antigen, showing the darker nodule to be comprised ofhuman cells, with the surrounding parenchyma being of murine origin.

FIG. 3 is a graph illustrating production of albumin from humanhepatocyte grafts (1×10⁶ cells) in four recipients carrying the Alb-uPAtransgene.

FIG. 4 is a Western blot of HA production in an Alb-uPA-positiverecipient post-transplant showing sustained signal intensity. HA—humanalbumin standard (50 ng); Con nontransplanted mouse serum control.

FIG. 5 is a photograph of a Western blot showing detection of humanalbumin (HA) produced from human hepatocytes in chimeric livers (samplesrepresent individual mice). Wild-type (−); transgenic (+) recipients.HA—human albumin standard; MS—nontransplanted mouse serum (negativecontrol).

FIG. 6 is a photograph of a Southern blot for determination of Alb-uPAzygosity from genomic DNA; a T/E ratio of 2 is characteristic ofhemizygous mice, while homozygotes have a ratio of 4.

FIG. 7 is a photograph of a Western blot showing long-term HA productionin transplant recipients hemizygous (+/−) or homozygous (+/+) for theAlb-uPA transgene. HA—human albumin standard; MS—nontransplanted mouseserum (negative control).

FIG. 8 is a graph showing a vertical scatterplot of quantified HAproduction from individual homozygous (closed circles) or hemizygous(open circles) recipient mouse serum samples. Median trend lines areshown for both groups.

FIG. 9 is a graph showing rising serum HCV RNA titers over the first 4-7weeks post inoculation in homozygous transgenic graft recipients afterinoculation with HCV-infected human serum. Each line represents serialtiters from an individual graft recipient.

FIG. 10A is a photograph of a gel showing detection of (+) strand RNA(upper panel) or (−) strand RNA (lower panel) by thermostable rTthreverse transcriptase RNA PCR protocol with strand-specific primers.Letter designations (A through J) are control samples and numberdesignations (1 through 10) represent individual RNA samples isolatedfrom the livers of ten homozygous mice which were transplanted and theninoculated with HCV-infected human serum. A, wild-type control mouse,nontransplanted, noninfected; B, heterozygous transplanted mouseinoculated with HCV; C, homozygous transplanted mouse, not inoculatedwith HCV; D, serum taken from an infected human; E, standard DNA ladder;F, binding of labeled probe to target DNA sequences generated from (+)strand (upper panel) or (−) strand (lower panel) viral RNA; G, mouseliver RNA (10 μg) doped with serum RNA from an HCV-positive human; H,mouse liver RNA (10 μg) doped with 10⁶ copies radioinert antisense(upper) or sense (lower) riboprobe; I, mouse liver RNA (10 μg) dopedwith 10⁶ copies radioinert sense (upper panel) or antisense (lowerpanel) riboprobe; J, riboprobes hybridized with 10 μg mouse liver RNA,all subsequent steps identical except addition of RNase. FIG. 10B is adilution series analysis.

FIG. 10B is a photograph of a gel of a dilution series analysis ofselected animals using the thermostable rTth reverse transcriptase RNAPCR protocol. Letter and number designations are the same as in FIG.10A.

FIG. 10 C is a photograph of a gel showing detection of (+) strand HCVRNA (upper panel), (−) strand HCV RNA (middle panel) or β-actin RNA(lower panel) by RNase protection assay. Control lanes are as designatedabove; mouse 10 was analyzed only by the RPA method. Letter and numberdesignations are the same as in FIG. 10A.

FIGS. 11A and 11B are photographs showing immunohistochemical analysisof control (FIG. 11A) and HCV infected (FIG. 11B) liver sections usingant anti-HCV antibody.

FIG. 12 is a photograph of a Western blot to detect Apo B100 in serum ofa chimeric Alb/uPA, transplanted animal (lane 2). Human serum (lane 1)and serum from Alb/uPA a non-transplanted animal (lane 3) served aspositive and negative controls, respectively.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

Before the present invention is described, it is to be understood thatthis invention is not limited to particular methodology, protocols, celllines, animal species or genera, constructs, and reagents described, assuch may, of course, vary. It is also to be understood that theterminology used herein is for the purpose of describing particularembodiments only, and is not intended to be limiting, since the scope ofthe present invention will be limited only by the appended claims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimits of that range is also specifically disclosed. Each smaller rangebetween any stated value or intervening value in a stated range and anyother stated or intervening value in that stated range is encompassedwithin the invention. The upper and lower limits of these smaller rangesmay independently be included or excluded in the range, and each rangewhere either, neither or both limits are included in the smaller rangesis also encompassed within the invention, subject to any specificallyexcluded limit in the stated range. Where the stated range includes oneor both of the limits, ranges excluding either or both of those includedlimits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methodsand materials are now described. All publications mentioned herein areincorporated herein by reference to disclose and describe the methodsand/or materials in connection with which the publications are cited.

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “and”, and “the” include plural referents unless thecontext clearly dictates otherwise. Thus, for example, reference to “aliver cell” includes a plurality of such liver cells and reference to“the non-human animal” includes reference to one or more non-humananimals and equivalents thereof known to those skilled in the art, andso forth.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.Further, the dates of publication provided may be different from theactual publication dates which may need to be independently confirmed.

DEFINITIONS

“Chimeric” as used herein (e.g., “chimeric animal” or “chimeric liver”)is meant to describe an organ or animal comprising xenogeneic tissues orcells. Of particular interest is a chimeric animal, wherein the animalis chimeric due to the presence of human hepatocytes engrafted in theanimal's liver.

By “immunocompromised” is meant that the animal can not mount a completeor significant immune response against the xenogeneic tissue or cells,e.g., any immune response of the host animal is such that it isineffective in rejection of the transplanted cells.

The term “transgene” is used herein to describe genetic material whichhas been or is about to be artificially inserted into the genome of amammalian, particularly a mammalian cell of a living animal.

By “transgenic animal” is meant a non-human animal, usually a mammal,having a non-endogenous (i.e., heterologous) nucleic acid sequencepresent as an extrachromosomal element in a portion of its cells orstably integrated into its germ line DNA (i.e., in the genomic sequenceof most or all of its cells). Heterologous nucleic acid is introducedinto the germ line of such transgenic animals by genetic manipulationof, for example, embryos or embryonic stem cells of the host animalaccording to methods well known in the art. A “transgene” is meant torefer to such heterologous nucleic acid, e.g., heterologous nucleic acidin the form of an expression construct (e.g., for the production of a“knock-in” transgenic animal) or a heterologous nucleic acid that uponinsertion within or adjacent a target gene results in a decrease intarget gene expression (e.g., for production of a “knock-out” transgenicanimal).

A “knock-out” of a gene means an alteration in the sequence of the genethat results in a decrease of function of the target gene, preferablysuch that target gene expression is undetectable or insignificant.Transgenic knock-out animals can be comprise a heterozygous knock-out ofa target gene, or a homozygous knock-out of a target gene. “Knock-outs”as used herein also include conditional knock-outs, where alteration ofthe target gene can occur upon, for example, exposure of the animal to asubstance that promotes target gene alteration, introduction of anenzyme that promotes recombination at the target gene site (e.g., Cre inthe Cre-lox system), or other method for directing the target genealteration postnatally.

A “knock-in” of a target gene means an alteration in a host cell genomethat results in altered expression (e.g., increased (including ectopic)or decreased expression) of a target gene, e.g., by introduction of anadditional copy of the target gene, or by operatively inserting aregulatory sequence that provides for enhanced expression of anendogenous copy of the target gene. “Knock-in” transgenics can comprisea heterozygous knock-in of the target gene or a homozygous knock-in of atarget gene. “Knock-ins” also encompass conditional knock-ins.

By “operably linked” is meant that a DNA sequence and a regulatorysequence(s) are connected in such a way as to permit gene expressionwhen the appropriate molecules (e.g., transcriptional activatorproteins) are bound to the regulatory sequence(s).

By “operatively inserted” is meant that a nucleotide sequence ofinterest is positioned adjacent a nucleotide sequence that directstranscription and translation of the introduced nucleotide sequence ofinterest.

The term “therapeutic agent” as used herein refers to any molecule,e.g., protein or small molecule, pharmaceutical compound, antibody,antisense molecule, ribozyme, and the like, useful in the treatment of adisease or condition, e.g., a liver condition, including, but notnecessarily limited to infection by HCV. For example, therapeutic agentsof the invention include molecules that inhibit, ameliorate, or relievesymptoms associated with viral infection, and in particular HCV.

The term “unit dosage form” as used herein refers to physically discreteunits suitable as unitary dosages for subjects (e.g., animals, usuallyhumans), each unit containing a predetermined quantity of agent(s) in anamount sufficient to produce the desired effect in association with apharmaceutically acceptable diluent, carrier or vehicle. Thespecifications for the novel unit dosage forms of the present inventionwill depend on a variety of factors including, but not necessarilylimited to, the particular agent employed and the effect to be achieved,and the pharmacodynamics associated with each compound in the host.

The terms “treatment”, “treating” and the like are used herein togenerally mean obtaining a desired pharmacologic and/or physiologiceffect. The effect may be prophylactic in terms of completely orpartially preventing a disease or symptom thereof and/or may betherapeutic in terms of a partial or complete cure for a disease and/oradverse effect attributable to the disease. “Treatment” as used hereincovers any treatment of a disease in a mammal, particularly a human, andincludes: (a) preventing the disease from occurring in a subject whichmay be predisposed to the disease but has not yet been diagnosed ashaving it; (b) inhibiting the disease, i.e., arresting its development;or (c) relieving the disease, i.e., causing regression of the disease.

Overview

The present invention is based on the development of a murine animalmodel having a chimeric liver with human hepatocytes, and which issusceptible to infection by human hepatitis C virus (HCV). The murineanimal model generally involves transplantation of human hepatocytesinto the liver of a transgenic mouse at an appropriate stage of thehost's development, preferably shortly after birth of the host. Withoutbeing held to theory, success in the development of the model is due atleast in part to the following: 1) use of a host having animmunodeficient background, thus avoiding immune destruction ofintroduced xenogenic (human) cells; 2) the use of a transgenic animalthat contains a transgene for urokinase linked to an albumin promoter,which is present in the homozygous state, thereby providing an ongoingpotent stimulus to hepatocyte growth and cellular division; and, 3)introduction of viable human hepatocytes into the host animal at anappropriate time in the hepatocyte life cycle and at an early stage ofthe host animal's development to provide for long-term survival ofeither large numbers and/or a high percentage of human cells in thehost.

To the best of the inventors' knowledge, the present invention for thefirst time provides a non-primate host for use as a model of HCVinfection that can be infected through the normal route of infection(e.g., by intravenous or intraperitoneal inoculation). This aspect ofthe invention is particularly important for use in the development ofanti-viral agents. Furthermore, the animal model of the invention doesnot require the use of pre-infected human hepatocytes, thus avoiding thehandling of infected tissue isolated from human donors or infecting thehuman hepatocytes in vitro prior to implantation.

Accordingly the invention features a chimeric animal as described above,as well as a method of producing a chimeric animal by transplantinghuman hepatocytes into the liver of an immunocompromised, albumin linkedurokinase transgene-bearing animal. In addition the invention featuresmethods of using the chimeric animal model described herein, includingmethods of identifying agents for treatment of infections by ahepatotrophic microbial pathogen.

In other aspects the invention features methods of using the non-humananimal model of the invention in assessing toxicity and evaluating drugsin modulation of levels of blood lipids.

The invention will now be described in more detail.

Host Animals

The host animal is generally a non-human, immunocompromised mammalhaving an increased production in the liver of urokinase-typeplasminogen activator (uPA) and in which human hepatocytes can beengrafted and maintained. Exemplary non-human animals upon which theanimal model of the invention can be based include, but are notnecessarily limited to, mice, rats, guinea pigs, hamsters, sheep, pigs,primates, and the like. In one embodiment, the host animal is of thegenus Rodentia, preferably a mouse.

In a preferred embodiment, the host animal is an immunocompromisedmouse, preferably an immunocompromised mouse transgenic forurokinase-type plasminogen activator (uPA), more preferably animmunocompromised mouse comprising a transgene that provides forliver-specific production of uPA (e.g., an Alb-uPA transgene, see, e.g.,Heckel et al Cell 62:447 (1990)). Mice suitable for use in the presentinvention can be produced from any of a variety of background strainsincluding, but not necessarily limited to, the strains C.B-17, C3H,BALB/c, C57131/6, AKR, BA, B10, 129, etc. The host animal may be eithermale or female.

Immunocompromised Background

As noted above, the host animal is preferably immunocompromised.Immunocompromised mammalian hosts suitable for implantation and havingthe desired immune incapacity are available. Alternatively, though lesspreferred, immunocompromised animals can be generated fromimmunocompetent animals by, for example, administration of one or morecompounds (e.g., cyclosporin) and other methods well known in the art.In general, the immunocompromised host can not mount a complete immuneresponse against the xenogeneic tissue or cells. Of particular interestare animals that are immunocompromised due to a genetic defect thatresults in an inability to undergo germline DNA rearrangement at theloci encoding immunoglobulins and T-cell antigen receptors. Also ofinterest are immunocompromised animals that have one or more geneticdefects that leads to significantly decreased numbers of or nodetectable functional T cells, B cells, and natural killer (NK) cellsrelative to normal.

Of particular interest are mice that have a homozygous mutation at thescid locus (scid/scid). The scid mutation is associated with adeficiency in DNA-dependent protein kinase catalytic subunit andprevents VDJ recombination in immunoglobulin and T-cell receptor genes.Animals homozygous for the scid mutation lack functionally recombinedimmunoglobulin and T-cell receptor genes and thus are deficient in bothT and B cell lineages. The scid/scid mutation is available or may bebred into a number of different genetic backgrounds, e.g., CB.17, ICR(outbred), C3H, BALB/c, C57B1/6, AKR, BA, B10, 129, etc. The inventioncan also take advantage of animals having the beige mutation (bg), whichis associated with a natural killer (NK) cell deficiency. In oneembodiment, mice are produced having both the scid mutation and the bgbeige mutation, resulting in an animal that does not mount an effectiveimmune response to allogeneic or xenogeneic cells or tissues introducedto the organisms.

Other exemplary immunocompromised host that are presently availableinclude transgenic mice genetically engineered to lack the recombinasefunction associated with RAG-1 and/or RAG-2 (e.g., commerciallyavailable TIM™ RAG-2 transgenic), to lack Class I and/or Class II MHCantigens (e.g., the commercially available C1D and C2D transgenicstrains), or to lack expression of the Bcl-2 proto-oncogene. Other micethat may be useful as recipients are NOD scid/scid; SGB scid/scid,bh/bh; CB.17 scid/hr; NIH-3 bg/nu/xid and META nu/nu. Transgenic mice,rats and pigs are available which lack functional B cells and T cellsdue to a homozygous disruption in the CD3F-gene. Immunocompromised ratsinclude HsdHan:RNU-rnu; HsdHan:RNU-rnu/+; HsdHan:NZNU-rnu;HsdHan:NZNU-rnu/+; LEW/HanHsd-rnu; LEW/HanHsd-rnu/+; WAG/HanHsd-rnu andWAG/HanHsd-rnu/+.

Transgenic Expression of Urokinase

As discussed above, the chimeric animal of the invention is also a“knock-in” transgenic for expression of urokinase-type plasminogenactivator (uPA). In one embodiment, the transgene is the Alb-uPAtransgene, which comprises a murine albumin enhancer/promoter, themurine uPA gene coding region, and the 3′ untranslated and flankingsequences of the growth hormone gene (Heckel et al. Cell 62:447-56(1990); Sandgren et al. Cell 66:245-56 (1991)). Preferably the animal ishomozygous, rather than heterozygous, for the urokinase-type plasminogenactivator transgene. The Alb-uPA transgene results in a lethal insult tohepatocytes that carry it, and also results in a high local(intrahepatic) concentration of urokinase, which in turn processeshepatocyte growth factor to its active form within the liver. Withoutbeing held to theory, viable allogeneic or xenogeneic cells introducedat an appropriate time in the development of an Alb-uPA transgenicanimal are stimulated to replicate in this environment. The donor cellsthus grow to “replace” the endogenous hepatocytes that die as a resultof the lethal insult of the transgene.

Isolation of Human Hepatocytes and Other Cells Suitable forTransplantation

Human hepatocytes for transplantation into the host animals are isolatedfrom human liver tissue by any convenient method known in the art. Ingeneral, the human hepatocytes may be fresh tissue (e.g., obtainedwithin hours of death), or freshly frozen tissue (e.g., fresh tissuefrozen and maintained at or below about 0° C.). Ideally, the cells usedare recently isolated (i.e., within 2 to 4 hours) from freshly obtainedhuman liver tissue. Human hepatocytes that are placed in a definedcryopreservation media may be stored for long periods of time (e.g., inliquid nitrogen) and thawed as required, thus permitting the developmentof banks of stored hepatocytes. In general, it is usually important thatthe isolation procedure and handling and storage protocol serve tominimize warm ischemia following cessation of blood flow to the liver(e.g., generally less than about 30 min to 60 min, preferably less thanabout 20 min to about 40 min) and to minimize cold ischemia that mayresult from storage (e.g., generally less than about 12 hr, usually lessthan about 1 hr to 2 hrs). In one embodiment, the human tissue isnormal, e.g., having no detectable pathogens, normal in morphology andhistology, and essentially disease-free). Usually the period of warmischemia exposure is not more than about 20-50 minutes.

The liver tissue can be dissociated mechanically or enzymatically toprovide a suspension of single cells, or fragments of intact humanhepatic tissue may be used. In a preferred embodiment, the hepatocytesare isolated from donor tissue by routine collagenase perfusion (Ryan etal. Meth. Cell Biol. 13:29 (1976)) followed by low-speed centrifugation.Hepatocytes can then be purified by filtering through a stainless steelmesh (e.g., 100 μm), followed by density-gradient centrifugation.Alternatively, other methods for enriching for hepatocytes can be used,e.g., fluorescence activated cell sorting, panning, magnetic beadseparation, elutriation within a centrifugal field, etc. The finalsuspension used for implantation generally comprises at least about50-75% hepatocytes, usually at least about 80-99% hepatocytes, generallywith viability by trypan blue exclusion of 80-99%,

In another embodiment, the cells to be transplanted are human stem cellsor hepatocyte precursor cells which, following transplantation into thehost animal's liver, develop or differentiate into human hepatocytessusceptible to HCV infection. In one specific embodiment, the human stemcells are obtained from human blood cord cells. Human blood cord cellsare not only a source for stem cell reconstitution of hepatocytes, butalso for reconstitution of the immune system (see, e.g., Verstegen etal. Blood. 91(6):1966-76 (1998)).

Transplantation of Human Hepatocytes or Other Suitable Cells into Hosts

The timing of the introduction of the donor hepatocytes into thetransgenic, immunocompromised host may be important to the production ofa chimeric liver populated with a number of human hepatocytes sufficientto render the chimeric liver susceptible to infection by a hepatotrophicpathogen and to support replication of the pathogen. This may beparticularly true where the hepatotrophic pathogen exhibits lowinfectivity and/or low replication rates (e.g., HCV). Where the animalis murine (e.g., a mouse), the host is ideally less than 10 days to 2weeks in age, and optimally about 7 to 10 days old, or less than orabout one week (i.e., less than or about 5 to 7 days old or younger), atthe time of transplantation. In general, the transplantation ispreferably carried out between about 8-10 days and 15 days of age. Thewindow for transplant can be widened to about 7-18 days of age to gainflexibility while maintaining good results. Without being held totheory, the timing of transplantation indicated herein is a compromisebetween excess technical mortality associated with very earlytransplantation (i.e., due to the small size of the animals) and thetime for maximal replicative stimulus (e.g., the number of celldivisions in the recipient liver that occur before transplant mayinfluence the success and extent of engraftment of the donor humancells). Furthermore, timing of transplantation is also important sincethe stimulus for liver cell repopulation provided by the transgenediminishes with time, and is generally depleted after the recipient ismore than about 6 weeks old (Rhim et al. (1994) Science 263:1149-52;about 10-12 weeks for homozygotes).

The human hepatocytes (or other suitable cell, e.g., hepatocyteprecursor or stem cell) can be transplanted using any suitable methodknown in the art. Preferably, the human hepatocytes are injectedintrasplenically, e.g., into the inferior splenic pole. Successfulengraftment can be monitored by conventional methods, e.g., by examiningthe levels of human liver-specific proteins in the host serum, e.g.,human serum albumin (HA), or human alpha-1 antitrypsin. The chimerichost can be used for experimentation (e.g., for infection with ahepatotrophic pathogen, to screen candidate agents, etc.) when suitable.Where the animal is to be infected with a hepatotrophic agent ofrelative low infectivity and/or low replicative capacity, the chimericanimal can be inoculated within about four to six weeks post-transplant,generally at about six weeks post-transplant, and may be as early asthree weeks post-transplant.

In general, the animal host develops human chimerism within its liversuch that the percentage of liver cells that are human liver cells arefrom at least about 20% to 50%, generally about 40% to 60% or more, andmay be optimized to 90% or more. The chimeric animal can be maintainedwith functional transplanted hepatocytes for at least several weeks,generally at least about 5 weeks, more usually at least about 12 weeksto 24 weeks, up to 8 months or more, and may be up to the lifespan ofthe host. Chimeric animals can be infected with a hepatotrophic pathogen(e.g., HCV), particularly a hepatotrophic pathogen having a host rangelimited to primates, particularly humans. Depending upon the nature ofthe pathogen, chronically infected chimeric hosts can be maintained fora period of weeks to months. For example, where the hepatotrophicpathogen is HCV, the chimeric animal can become chronically infectedwith HCV (e.g., chronically infected) and maintain an active HCVinfection for a period of at least about 5 weeks, generally at leastabout 14 weeks to about 20 weeks or more, up to about 35 weeks or more,and may be for the lifespan of the host.

The viral load of the infected host can be established such that it issimilar to the viral load of an infected human. For example, where thepathogen is HCV, the host animal can support infection at a level offrom about 10³ or about 10⁴ to about 10⁶ viral particles/ml serum,generally from about 10³ to about 10⁷ viral particles/ml serum.

The viral load of the infected host over time is substantiallyconsistent, chronic, and stable, e.g., the number of viral particlesthat can be isolated from the infected. untreated host's serum does notradically fluctuate between weekly sampling periods, e.g., anHCV-infected host of the invention that contains a high number of HCVviral particles per mL of serum at a first sampling time is positive forHCV infection at subsequent sampling times and generally has the same orsimilar high level of HCV particles per mL of serum, once stableinfection is established in the host, generally within about 2 to 4weeks post-infection. In general, the viral load of the infected hostdoes not fluctuate radically and so allows assessment of the effect of acandidate antiviral agent, e.g., the viral titer is chronic andreasonably consistent.

Screening Assays

The chimeric animal of the invention can be used in a variety of otherscreening assays. For example, any of a variety of candidate agentssuspected of causing or contributing to hepatic disease, as well as theappropriate antagonists and blocking therapeutic agents, can be screenedby administration to the chimeric animal and assessing the effect ofthese agents upon function of the engrafted human cells.

In one embodiment of particular interest, the animal model of theinvention can be used to identify candidate agents that, for example,inhibit or prevent infection by, replication of, or disease symptomscaused by a hepatotrophic pathogen (e.g., bacteria, virus, parasite,especially a hepatotrophic virus such as HCV). Although the examplesprovided herein generally involve the use of chimeric murine hosts witha single hepatotrophic pathogen, the invention can also be used toidentify a single candidate agent or a cocktail of candidate agentshaving activity against infection by two or more hepatotrophic agents.

“Candidate agents” is meant to include synthetic, naturally occurring,or recombinantly produced molecules (e.g., small molecule; drugs;peptides; antibodies (including antigen-binding antibody fragments,e.g., to provide for passive immunity) or other immunotherapeuticagents; endogenous factors present in eukaryotic or prokaryotic cells(e.g., polypeptides, plant extracts, and the like)); etc.). Ofparticular interest are screening assays for agents that have a lowtoxicity for human cells.

Candidate agents encompass numerous chemical classes, though typicallythey are organic molecules, preferably small organic compounds having amolecular weight of more than 50 and less than about 2,500 daltons.Candidate agents comprise functional groups necessary for structuralinteraction with proteins, particularly hydrogen bonding, and typicallyinclude at least an amine, carbonyl, hydroxyl or carboxyl group,preferably at least two of the functional chemical groups. The candidateagents often comprise cyclical carbon or heterocyclic structures and/oraromatic or polyaromatic structures substituted with one or more of theabove functional groups. Candidate agents are also found amongbiomolecules including, but not limited to: peptides, saccharides, fattyacids, steroids, purines, pyrimidines, derivatives, structural analogsor combinations thereof.

Candidate agents are obtained from a wide variety of sources includinglibraries of synthetic or natural compounds. For example, numerous meansare available for random and directed synthesis of a wide variety oforganic compounds and biomolecules, including expression of randomizedoligonucleotides and oligopeptides. Alternatively, libraries of naturalcompounds in the form of bacterial, fungal, plant and animal extractsare available or readily produced. Additionally, natural orsynthetically produced libraries and compounds are readily modifiedthrough conventional chemical, physical and biochemical means, and maybe used to produce combinatorial libraries. Known pharmacological agentsmay be subjected to directed or random chemical modifications, such asacylation, alkylation, esterification, amidification, etc. to producestructural analogs.

Screening of Candidate Anti-HCV Agents

In one embodiment, the animal model of the invention is used to identifyagents that ameliorate symptoms caused by viral hepatitis, and morespecifically by HCV infection and/or to more directly affect apathogenic mechanism of the infecting virus, e.g., inhibit viralinfection, decrease viral replication, or otherwise disrupt the cycle ofviral propagation. In general, the candidate agent is administered tothe animal model of the invention, and the effects of the candidateagent assessed relative to a control (e.g., relative to an uninfectedanimal, relative to an HCV-infected animal treated with an agent havinga known anti-HCV effect (e.g., IL-2α), and the like). For example, thecandidate agent can be administered to an HCV-infected animal of theinvention, and the viral titer of the treated animal (e.g., as measuredby RT-PCR of serum samples) compared to the viral titer of the animalprior to treatment and/or to a control, untreated HCV-infected animal.In general, a detectable and significant decrease in viral titer of aninfected animal following treatment with a candidate agent is indicativeof antiviral activity of the agent.

The candidate agent can be administered in any manner desired and/orappropriate for delivery of the agent in order to effect a desiredresult. For example, the candidate agent can be administered byinjection (e.g., by injection intravenously, intramuscularly,subcutaneously, or directly into the tissue in which the desired affectis to be achieved), orally, or by any other desirable means. Normally,the in vivo screen will involve a number of animals receiving varyingamounts and concentrations of the candidate agent (from no agent to anamount of agent that approaches an upper limit of the amount that can bedelivered successfully to the animal), and may include delivery of theagent in different formulations and routes. The agents can beadministered singly or can be combined in combinations of two or more,especially where administration of a combination of agents may result ina synergistic effect.

The activity of the candidate agent can be assessed in a variety ofways. For example, where the host animal is infected with ahepatotrophic pathogen (e.g., HCV, etc.), the effect of the agent can beassessed by examining serum samples for the presence of the pathogen(e.g., titer, as in viral titer) or markers associated with the presenceof the pathogen (e.g., a pathogen-specific protein or encoding nucleicacid, etc.) Qualitative and quantitative methods for detecting andassessing the presence and severity of viral infection are well known inthe art. In one embodiment, the activity of an agent against HCVinfection can be assessed by examining serum samples and/or tissuesections for the presence of a virus (e.g., HCV by RT-PCR, etc.). Inanother embodiment, the activity of an agent against viral infection canbe assessed by examining serum samples for the presence of viral nucleicacid (e.g., HCV RNA). For example, HCV RNA can be detected using, forexample, reverse transcriptase polymerase chain reaction (RT-PCR),competitive RT-PCR or branched-DNA (bDNA) assay, detection ofnegative-strand RNA (the replicative intermediate of HCV) by RT-PCR, orsequencing of viral RNA to detect mutation/shift in the viral genome(“quasispecies evolution”) with therapy. Alternatively or in addition,the host liver may be biopsied and in situ RT-PCR hybridizationperformed to demonstrate directly any qualitative or quantitativealterations in the amount of viral particles within tissue sections.Alternatively or in addition, the host can be euthanized and the liverexamined histologically for signs of infection and/or toxicity caused bythe agent.

Identified Agents

The compounds having the desired pharmacological activity may beadministered in a physiologically acceptable carrier to a host fortreatment. The therapeutic agents may be administered in a variety ofways, orally, topically, parenterally e.g. subcutaneously,intraperitoneally, intravascularly, by inhalation, etc. Depending uponthe manner of introduction, the compounds may be formulated in a varietyof ways. The concentration of therapeutically active compound in theformulation may vary from about 0.1-100 wt. %.

The pharmaceutical compositions can be prepared in various forms, suchas granules, tablets, pills, suppositories, capsules, suspensions,salves, lotions and the like. Pharmaceutical grade organic or inorganiccarriers and/or diluents suitable for oral and topical use can be usedto make up compositions containing the therapeutically-active compounds.Diluents known to the art include aqueous media, vegetable and animaloils and fats. Stabilizing agents, wetting and emulsifying Agents, saltsfor varying the osmotic pressure or buffers for securing an adequate pHvalue, and skin penetration enhancers can be used as auxiliary agents.

Vaccine Development

With some modifications, the animal model of the invention can also beused to screen candidate vaccines for their ability to prevent orameliorate infection by a hepatotrophic pathogen. In general, a“vaccine” is an agent that, following administration, facilitates thehost in mounting an immune response against the target pathogen. Thehumoral, cellular, or humoral/cellular immune response elicited canfacilitate inhibition of infection by the pathogen against which thevaccine is developed. Of particular interest in the present inventionare prophylactic vaccines that elicit a protective immune response thatinhibits infection by and/or intrahepatic replication of a hepatotrophicpathogen, e.g., a microbial, viral, or parasitic pathogen, particularlya viral pathogen, e.g., HCV. Also of interest are therapeutic vaccineswhich provide protection through provision of passive immunity orrapidly upregulated specific active immunity (e.g., anti-HCVimmunoglobulin, and the like).

In this embodiment of the invention, the immune system of theimmunocompromised chimeric animal is reconstituted using, for example,stem cells, peripheral blood mononuclear cells (PBMCs), blood cordcells, hematopoietc cells, or other suitable cells of human origin toprovide for a human immune system in the animal. Methods for isolatinghuman immune cells and reconstitution of the immune system of animmunocompromised animal, e.g., a mouse with an human immune system arewell known in the art (see, e.g., Nature 335:256-59; Proc. Natl. Acad.Sci. USA 93(25):14720-25). In one embodiment, the human immune cells areobtained from the same donor as the human hepatocytes used in theproduction of the chimeric liver. In one embodiment, the human immunecells are introduced into the host according to methods well known inthe art, e.g., by intraperitoneal injection.

Screening for an effective vaccine is similar to screening methodsdescribed above. In short, the candidate vaccine is administered to thechimeric animal prior to inoculation with the hepatotrophic pathogen.The candidate vaccine is generally administered by providing a singlebolus (e.g., intraperitoneal or intramuscular injection, topicaladministration, or oral administration), followed by one or more boosterimmunizations. The induction of an immune response can be assessed byexamining B and T cell responses that are specific for the antigenaccording to methods well known in the art. The immunized animal is thenchallenged with the hepatotrophic pathogen; normally several immunizedanimals are challenged with increasing titers of the pathogen. Theimmunized animals and non-immunized control animals are then observedfor development of infection, and the severity of infection assessed(e.g., by assessing the titer of the pathogen present, examining humanhepatocyte function parameters as described above, etc.). Vaccinecandidates that provide for a significant decrease in infection by thepathogen and/or a significant decrease in the severity of disease thatresults post-challenge are identified as viable vaccines.

Other Uses

Uses of the chimeric animal of the invention that are variations upon orin addition to those described above will be readily apparent to theordinarily skilled artisan upon reading of the present specification

Infectious Disease Diagnosis

For example, the chimeric animal can be infected, preferably chronicallyinfected, with a hepatotrophic agent, and used as a source from whichthe agent can be isolated. This use of the chimeric animal of theinvention is particularly useful where, for example, isolation of thepathogen requires biopsy from a human subject or is difficult to obtainin useful amounts; the pathogen cannot be readily cultured in vitro;culturing of the pathogen in vitro (e.g., growth in broth culture or incultured cells) leads to changes in the pathogen that may affects itspathogenicity and/or clinical relevance; etc. In general, the chimericanimal is inoculated with the isolated pathogen by an appropriate route(e.g., by intravenous, intramuscular, intraperitoneal, or oraladministration), preferably by a route of infection that best correlateswith the natural route of infection in human disease. After the pathogenestablishes infection of the human hepatocytes, and after a sufficientamount of time has passed to allow replication of the pathogen, thepathogen is isolated from the infected chimeric animal by an appropriatemethod (e.g., isolation from a blood sample, from liver, etc.).

Liver disease diagnosis. The chimeric animal can also be used in thecourse of diagnosis of liver disease in a human. For example, where thepatient suffers from a liver disease of unknown origin or wherediagnosis without culturing of the pathogen is not definitive, a samplesuspected of containing the causative agent can be isolated from thepatient (e.g., from the patient's serum or from a liver biopsy). Thesample can be enriched for the suspected agent, fractionated, orotherwise processed to provide it in an administrable form, andadministered to the chimeric animal. The chimeric animal can then beevaluated to assess the effect of administration of the sample upon theengrafted human hepatocytes. The effect upon the human hepatocytes canbe accomplished by, for example, isolation and examination of serumsamples from the chimeric animal, e.g., to assess function of theengrafted human hepatocytes, and/or to detect a pathogen in the animal'sserum, e.g., to detect the presence of HCV or other microbial pathogen).The human hepatocytes can also be examined histologically to determinethe effect of the patient sample.

Screening using patient samples. The invention can also be adapted toprovide for diagnosis and rationale therapy designed on anindividualized basis. For example, human hepatocytes obtained by biopsyof a patient (e.g., percutaneous needle biopsy) can be used to producethe chimeric murine host. This chimeric murine host can then be used toevaluate the hepatotrophic pathogen infecting the patient, assess thepathogen's susceptibility to therapeutic agents, and to assess thepotential toxicity of the patient's hepatocytes to such therapy. Thusthe invention can be designed to facilitate tailoring of therapies mosteffective against an individual's specific hepatotrophic pathogencomplement (e.g., against one or more infecting hepatotrophicpathogens).

Screening for agents that reduce blood lipids. The invention can also beadapted as a system for evaluation of potential therapies of humanatherosclerotic vascular (including cardiovascular) disease.Atherosclerosis is the primary cause of heart attack and stroke in theWestern world and ultimately is responsible for nearly half themortality in Canada (Ross (1993) Nature 362: 801-809). A positivecorrelation between high levels of low density lipoprotein (LDL) andatherosclerosis has been realized for several decades (Brown et al. Ann.Rev. Biochem. 52: 223-261 (1983)). LDL is derived from very low densitylipoprotein (VLDL) in the circulatory system by virtue of a complexseries of reactions involving hydrolases, and transfer of lipids andapoproteins among lipoproteins (Fielding et al. (1996) In “Biochemistryof Lipids, Lipoproteins and Membranes”, (D. E. Vance and J. E. Vanceeds.) pp 495-516, Elsevier Science Publishers, Amsterdam). VLDL issecreted into the blood stream via an intricate secretory pathway(Gibbons, Biochem. J., 268: 1-13 (1990); Dixon et al. J. Lipid Res. 34:185-1 (1993); Sniderman et al. Arterioscler. Thromb. 13: 629-636 (1993);Yao et al. Biochim. Biophys. Acta. 1212:152-166 (1994); Davis et al.(1996) In “Biochemistry of Lipids, Lipoproteins and Membranes” (D. E.Vance and J. E. Vance eds.) pp. 473-493, Elsevier, Amsterdam; Innerarityet al., J. Biol. Chem. 271: 2353-2356 (1996)).

Apolipoprotein (apo) B is a major apoprotein of VLDL and, is the soleapoprotein of LDL. A relationship between high levels of apo B in plasmaand the risk of cardiovascular disease has been identified (Sniderman etal., Proc. Natl. Acad. Sci. USA 77: 604-608 (1980)). Regression ofcoronary artery disease has been observed in men aggressively treatedwith lipid lowering drugs that also cause a decrease in plasma apo B(Brown et al., N. Engl. J. Med. 323: 1289-98 (1990)). Thus, there is apositive link among the secretion of apo B from the liver, the ambientconcentration of apo B-containing lipoproteins in plasma and theincidence of atherosclerosis. Apo B is a large glycoprotein that isparamount in the assembly and secretion of lipids, includingtriglyceride and cholesterol of both dietary and endogenous origin. Inaddition, apo B is important in the intravascular transport andreceptor-mediated uptake and delivery of distinct classes oflipoproteins. The importance of apo B thus spans a range of functions,from the absorption and processing of dietary lipids to the regulationof circulating lipoprotien levels. This latter property underlies itsrelevance in terms of atherosclerosis susceptibility.

Two forms of apo B exist in mammals. Apo B100 represents the full-lengthprotein containing 4536 amino acids and is the exclusive formsynthesized in human liver (Young, Circulation 82: 1574-1594 (1990)).Apo B100 is the major protein constituent of LDL and contains the domainrequired for interaction of this lipoprotein species with the LDLreceptor (Young, 1990, supra). In addition, Apo B100 contains anunpaired cysteine residue, at position 4326, which mediates a covalentinteraction with apo(a) and thereby generates another distinctatherogenic lipoprotein, referred to as Lp(a) (Callow et al., Proc.Natl. Acad. Sci. USA 91: 2130-2134 (1994); McCormick et al., J. Biol.Chem. 271: 28294-28299 (1996)). The small intestine of all mammals, aswell as the liver of certain species, synthesize apo B48. In humans, apoB48 circulates in association with chylomicrons and chylomicronremnants, and these particles, by virtue of their content of apo E arecleared by a distinct receptor referred to as the LDL-receptor relatedprotein (Herz et al. Curr. Opin. Lipidol 6: 97-103 (1995).

In humans, current evidence indicates that susceptibility toatherosclerosis is most likely due to unfavorable combinations ofmutations affecting genes in several pathways, but our knowledge aboutwhich genes are involved is limited (Ross, 1993, supra). Due to theability to introduce or mutate genes, the mouse has become the mostcommon experimental animal model for atherosclerosis research. Wildtypemice on a chow diet do not get atherosclerosis. Three ways to induceatherosclerosis in mice are: diet-induced (Paigen et al., Proc. Natl.Acad. Sci. USA 84: 3763-3767 (1987)), apo E deficiency-induced(Piedrahita et al., Proc. Natl. Acad. Sci. USA. 89: 4471-4475 (1992);Plump et al., Cell 71: 343-353 (1992); Zhang et al., Science 258:468-471 (1992)), and LDL receptor-deficiency induced (Ishibashi et al.,J. Clin. Invest. 92: 883-893 (1993)). Thus murine transgenic modelsexpressing human genes involved in lipoprotein metabolism haveincreasingly served as small mammalian models where the spectra of bothnormal and pathologic human serum lipid profiles can be simulated, andin several instances have demonstrated the formation of atheroscleroticlesions. For example, the atherosclerotic lesions in apo E-deficientmice have been well characterized, and they resemble human lesions intheir sites of predilection and progression to the fibroproliferativestage. These mouse models of atherosclerosis are being used to identifygenes which modify atherosclerosis susceptibility and in the developmentof antiatherogenic therapies.

The animal model of the present invention can likewise serve as ananimal model for hyperlipidemia and artherosclerosis, and can be used toidentify candidate agents having activity in reducing the risk of suchdiseases (e.g., useful in prophylactic treatment) or in treating suchdiseases (e.g., by lowering blood lipids). Study of serum from thechimeric, transgenic animal model of the invention has demonstrated thepresence of the human lipoprotein apoB100. Since this molecule has beenestablished as an important etiologic factor in the development of humanatherosclerotic vascular disease, screening for agents that affectapoB100 levels (either quantitatively or qualitatively) can serve toidentify agents that can modulate blood lipid levels and thus providetherapy for disease in humans. The positive control for such screeningassays can be human serum and, with non-transplanted homozygous Alb/uPAmouse serum serving as the negative control.

Methods for detection of apoB100 are well known in the art. Generally,the assay involves detection of formation of antibody-apoB100 complexesfollowing contacting a biological sample from the animal (e.g., blood,serum, plasma, and the like) with an antibody that specifically bindsapoB100. Detection of formation of antibody-apoB100 complexes can beaccomplished in a variety of ways (e.g., Western blot, dot blot, RIA,and the like).

Assessing Toxicity of an Agent.

The chimeric animal model of the invention can also be used to screencompounds for toxicity to liver cells, including small moleculetherapies for the treatment of liver disorders or for the treatment ofany non liver specific human diseases. In general, any compound can beadministered to evaluate its toxicity to liver cells. For example,evaluation of an important putative therapy for cancer can be firstscreened for liver toxicity in the animal model of the invention.Function of the engrafted human liver cells can be assessed as describedabove (e.g., by assessing levels of human serum albumin, or alpha-1antitrypsin in the host serum). Injury to liver cells can be assessed byassay of liver specific enzymes in the serum (ALT—alanineaminotransferase), in conjunction with histological assessment forevidence of injury to human cells in the liver. In short, assays toassess liver toxicity can be either functional, histological, or both.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the present invention, and are not intended to limit thescope of what the inventors regard as their invention nor are theyintended to represent that the experiments below are all or the onlyexperiments performed. Efforts have been made to ensure accuracy withrespect to numbers used (e.g. amounts, temperature, etc.) but someexperimental errors and deviations should be accounted for. Unlessindicated otherwise, parts are parts by weight, molecular weight isweight average molecular weight, temperature is in degrees Centigrade,and pressure is at or near atmospheric.

Example 1 Production of Alb-uPA Transgenic Mice

To generate an Alb-uPA transgenic mouse tolerant to human tissue grafts,mice heterozygous for the transgene (strain TgN(Alb1Plau)144Bri (TheJackson Laboratory)) were crossed with animals from a C.b-17/SCID-beigelineage (strain C.b-17/GbmsTac-scid-bgN7 (Taconic Farms), homozygous).Through a series of backcrosses, the SCID-beige trait was bred tohomozygosity as confirmed by quantification of total serum IgG using asandwich ELISA technique to detect mouse IgG according to methods wellknown in the art. Quantification of IgG was calculated from a standardcurve prepared on each plate using a mouse IgG standard (Cappel).“Leakiness” of the SCID-beige trait was defined as >1% of normal serumIgG (Bosma et al. Ann. Rev. Immun. 9:323 (1991)); animals with serum IgGlevels above this cutoff were euthanised. At each step, animals carryingthe Alb-uPA transgene were identified by PCR analysis of genomic DNAextracted from tail biopsies, using two 18-mer primers that amplify a151 bp product from the 3′ UTR of the transgene construct (JacksonLaboratories technical support). Although the homozygous Alb/uPA traithas been previously associated with a high perinatal mortality ratesecondary to bleeding complications and liver failure (Heckel et al.Cell 62:447 (1990)), we found that in our scid/bg/Alb-uPA animal colonyneonatal mortality was approximately 30%. The colony providing animalswas developed initially with heterozygous breeders, but with moderateneonatal mortality in homozygous mice, the colony was evolved tocompletely homozygous. Animals were housed in virus/antigen-freeconditions, and were cared for in accordance with the guidelinesestablished by the Canadian Council on Animal Care (1993). All animalexperiments describe Herein were performed with approval from theUniversity of Alberta Animal Welfare Committee.

Human hepatocytes for transplantation were obtained with approval fromthe University of Alberta Faculty of Medicine Research Ethics Board.Segments of human liver tissue (15-20 cm³) obtained at laparotomy wereperfused with ice-cold Ca/Mg-free PBS containing 0.5 mM Na₂EDTA.Prominent perfusing vessels were cannulated and the tissue was perfusedfor 30 minutes with recirculating carrier solution (35 mM NaCl, 3.5 mMKCl, 2.5 mM CaCl₂, 50 mM HEPES, pH 7.6) containing 0.38 mg/mL LiberaseCI collagenase (Boeringer-Mannheim) (Ryan et al. Surgery 113:48 (1993);Seglen et al. Meth. Cell Biol. 13:29 (1976)). Hepatocytes were filteredthrough 100 μm stainless steel mesh, purified by density-gradientcentrifugation (Percoll, density 1.04 g/mL; Sigma) at 400 g for 5minutes, and washed twice in ice-cold HBSS prior to suspension inBelzer-University of Wisconsin solution (DuPont) at 0° C. for short-termstorage prior to transplantation. Cell counts and viability wereconfirmed by trypan blue exclusion prior to transplantation; finalviability was routinely >80%.

In initial experiments, animals homozygous for the SCID trait andheterozygous for the Alb-uPA transgene were crossed, and 7 day-oldprogeny were transplanted with 1×10⁶ freshly isolated viable humanhepatocytes. Transplantation was accomplished by intrasplenic injection.Intrasplenically injected hepatocytes rapidly translocate to the livervia the portal venous system and engraft into the parenchyma surroundingterminal portal venules (Ponder et al. Proc. Natl. Acad. Sci. USA88:1217 (1991); Gupta et al. Transplantation 50:472 (1990)). Since themortality associated with intrasplenic injection is minimal, the spleenwas selected as the optimal site for implantation. Accordingly,offspring (5-17 days old) were anesthetized with Halothane/O₂, and asmall left flank incision was made. Under operating magnification, 1×10⁶viable hepatocytes were injected into the inferior splenic pole with a27 g butterfly injection set (Becton-Dickinson), and a single steriletitanium clip was placed across the injection site for hemostasis. Thespleen was returned to the abdomen, and the flank incision was closed intwo layers.

Since the production of albumin is an exclusive property of hepatocytes(Clement et al, Hepatology 4:373 (1984); Gunsalas et al. Nature Medicine3:48 (1997)), detection of human albumin (HA) in serum samples byselective immunoprecipitation and Western blotting was employed as anindicator of graft cell function. Recipient mice were initially sampledby jugular venous puncture at four weeks post-transplant, and at weeklyintervals thereafter. Aliquots of mouse serum (20 μl) were incubatedwith an anti-human albumin monoclonal antibody (Clone HSA-9; Sigma), andantigen-antibody complexes were precipitated with protein G-agarose(Boehringer-Mannheim). Immunoprecipitates were heated for 5 minutes at98° C. in SDS buffer containing 0.2 M dithiothreitol, separated bySDS-polyacrylamide gel electrophoresis and transferred tonitrocellulose. Western blots were prepared in standard fashion (Coliganet al. Current Protocols in Immunology (Wiley, New York, 1997), vol. 2,chap. 8.10.7) using a second anti-human albumin monoclonal antibody(Clone HSA-11; Sigma) conjugated to biotin as the primary. Astreptavidin-HRP conjugate (Pierce) was employed as the secondary, andchemiluminescent reagents (Pierce) were used for signal detection.

A strong HA signal was demonstrated in the serum of 4/7 transplantedlittermates, indicating the presence of significant numbers offunctional human hepatocytes; subsequent genotype analysis revealed thatall HA-positive animals carried the Alb-uPA transgene, whereas all theanimals negative for HA were also negative for the transgene. Clear HAbands were detected as early as two weeks post-transplant, with anincrease in intensity over the 4-6 week timepoints, suggesting vigorousexpansion of the primary cell grafts (FIG. 1). These findings indicatedthat the microenvironment within the Alb-uPA liver was sufficient tostimulate human hepatocytes to begin rapid proliferation, and that therewas the potential to support the establishment of long-term humangrafts.

To confirm proliferation and estimate the extent of replacement ofmurine parenchyma with human-derived cells, formalin fixed, paraffinembedded sections of recipient livers were obtained at various timesafter transplantation and immunostained with a monoclonal antibodyspecific for human hepatocytes. Segments of mouse liver were fixed in10% formalin and embedded in paraffin. Sections 5 u thick were stainedwith hematoxylin and eosin (H&E) in standard fashion. Selected sectionswere treated with an endogenous avidin/biotin blocking kit (ZymedLaboratories, Inc.) and immunostained with a monoclonal anti-humanhepatocyte antibody (DAKO, 1:20 dilution); bound antibody was detectedusing the Super Sensitive Immunodetection System (BioGenex)

The results are shown in FIGS. 2A-2F. In animals carrying the transgene,clusters of cells staining positive with the anti human hepatocyteantibody (darkly stained cells) were scattered uniformly throughout thehost liver at two weeks post-transplant, comprising an estimated 2-3% ofall hepatocytes. At four weeks the percentage of positive-staining cellshad increased, covering from 20 to 60% of the total surface area ofindividual sections. The interface between human and mouse cells wasdistinct, with cords of human cells extending into the surroundingmurine parenchyma. Individual human cells maintained a normal appearanceand developed sinusoidal architecture, although portal triad structureswere notably absent from the regenerating nodules. This latterobservation was not unexpected, since human-derived nodules are theresult of clonal expansion of individual hepatocytes (Sandgren et al.Cell 68:245 (1991)). These nodules would contain no bile duct orendothelial precursor cells; such structures would be host-derived andtherefore marginalized around proliferating human tissue.

Analysis of human hepatocyte graft function. Two different serum-basedassays were used to evaluate the human hepatocyte graft in our chimericmice. The first assay is a dot blot assay measuring human serum albumin;the second assay is an ELISA assay measuring human alpha-1 antitrypsin(hAAT). The same mice were assayed at 6 and 12 weeks post transplant.

The dot blot assay was performed by diluting 2 μl of sample or standardinto 40 μl of reducing buffer and heat for 5 min at 100 degrees(standards=known amounts of human albumin in blank mouse serum). A 2 μlvolume of solution was blotted onto Nitrocellulose membrane and allowedto dry for 15 min. The membrane was soaked in Western Transfer Solutionfor 10 min, and then blocked with 3% TBST for 1 hour. The membrane waswashed, and monoclonal antibody applied to reduced human albumin at1:5000 for 2 hours. After washing, horseradish-peroxidase-streptavidinat 1:10000 was applied for 1 hour, followed by washing and developingwith ECL-PLUS chemiluminescent solution. The membrane is then read usinga phosphoimager. The standard curve was plotted using standards and usethis curve to calculate sample values.

The ELISA was performed by coating plates with polyclonal goat-anti-hAATantibody at 1:1000 overnight, washing, and then blocking with TBST/milkbuffer overnight. After washing, the standards and samples, dilutedappropriately in milk buffer, were applied and incubate for 2 hours atRT. After washing, secondary antibody linked to HRP at 1:300 (diluted inmilk buffer) was applied and incubated for 2 hours at RT. After washing,TBMD substrate was added, and the reaction stopped at 5 minutes byaddition of 1 M H₂SO₄. The plate was read at 450 nm. A standard curvewas plotted using standards and this curve used to calculate samplevalues.

The table below shows the results obtained with each assay.

Age post- DOT BLOT ELISA Mouse transplant (μg/ml human albumin) (μg/mlAAT) DfRP (HOMO)  6 weeks 2283 244 12 weeks 1717 173 CfRP (HOMO)  6weeks 385 86 12 weeks 594 74 CfRM (HOMO)  6 weeks undetectable 0.7 12weeks dead dead BfLM (HOMO)  6 weeks 154 17 12 weeks 382 40 BmLP (HOMO) 6 weeks 608 45 12 weeks 767 96 AfRP (HETERO)  6 weeks{grave over ( )}99 7 12 weeks undetectable 1 AmLM (Hetero)  6 weeks 200 14 12 weeksundetectable 2

HOMO indicates animal is homozygous for the transgene; HETERO indicatesthe animal is heterozygous for the transgene. Both assays show ingeneral the trends are the same, showing a much higher production ofhuman-derived proteins in the homozygote for the uPA transgene comparedwith the heterozygotes.

Conclusion. This example demonstrates successful transplantation of theimmunocompromised, scid/bg/Alb-uPA mice with human hepatocytes.

Example 2 Persistence and Proliferation of Engrafted Human Hepatocytes

To determine the long-term outcome of initial successful engraftment andproliferation, a second litter of 8 animals was transplanted in similarfashion. The hepatocytes available for use at the time of thisexperiment were obtained from a patient who was a chronic carrier ofhepatitis B virus. The patient exhibited both a positive serum HBsAglevels and negative serum HBV DNA, indicating a chronic carrier statewithout active viral replication (Davis, South. Med. J. 90:866 (1997)).

Two randomly selected animals were sacrificed at 4 weeks for histologicanalysis, and the remaining 6 animals were followed at weekly intervals.Serum samples were subjected to Western blot as described above, and theHA bands from Western blots quantified using image analysis software andband densitometry (Umax Astra 1200S scanner and VistaScan DA v.1.2.2imaging software (UMAX Copr, Fremont, Calif.). Quantification of HApeaks was performed using NIH Image 1.60/fat software (NationalInstitute of Health), and normalized to a 50 ng HA standard present oneach blot.

Again, initial graft proliferation was seen only in the 4 animals whichcarried the transgene. In these animals, HA signals remained nearmaximal to 8 weeks at which point two distinct patterns of graftfunction emerged (FIG. 3; Mouse 3, open square; Mouse 4, closedtriangle; Mouse 5, open circle; Mouse 6, closed circle).

In three animals graft function began to slowly decline, with extinctionof the HA signal at 10, 15 or 16 weeks. In contrast, the fourthtransgenic animal (mouse no. 6) showed maximal HA production at allmeasured timepoints (FIGS. 3 and 4), indicating stable engraftment ofhuman hepatocytes. Sustained graft function repeatedly occurred inapproximately 25% of animals carrying the transgene. The proliferativesignal for the transplanted hepatocytes is likely dependent on overallexpression of the transgene, and is reduced as host-derived hepatocytesspontaneously delete the transgene.

In order to assess whether the transplanted mice supported the HBVinfection of the HBV-infected, transplanted cells, serum samples fromall transplanted mice were screened for hepatitis B surface antigen(HBsAg) production by sandwich ELISA. Aliquots of serum (20 μl) weretested for presence of HBsAg using a sandwich ELISA kit (HeprofileHbsAg; ADI Diagnostics) with plate analysis performed using a DynatechMRX microplate spectrophotometer (Dynex). Both positive and negativehuman serum controls, as well as negative murine serum controls wereincluded in assays.

The results are summarized in Table 1. Negative human and mouse serumcontrols range from 0.04-0.05 absorbance units; positive human controlsrange from 0.30-0.40 absorbance units.

TABLE 1 Analysis of serum markers of hepatitis B infection followingtransplantation of mice with HBV-infected human hepatocytes. AlbuPA HAEx- HBsAg Level Post-Transplant* Geno- pression 6 8 10 12 16 Mouse typePattern wk wk wk wk wk 1 − Absent ND 0.04 0.04 0.04 ND 2 − Absent 0.040.03 ND 0.02 ND 3 + Transient 0.04 0.03 0.08 0.05 ND 4 + Transient 0.120.04 0.07 0.04 ND 5 + Transient 0.04 0.03 ND 0.04 ND 6 + Persistent 0.130.13^(†) 3.18^(†) 3.78^(†) 3.44^(†) Key: HA—human albumin; ND—not done;*HBsAg levels expressed as absorbance units. ^(†)Samples positive forHBV DNA by PCR analysis.

As expected, control (Alb-uPA negative, nos. 1-2) mice had undetectableHBsAg levels and the three transgenic animals with transient graftfunction showed only sporadic minimal increases during weeks 6-12.However, the transgenic mouse with the pattern of sustained graftfunction (mouse no. 6) demonstrated clearly elevated levels at all timepoints measured, with an abrupt increase after 8 weeks to persist wellwithin the range of HBsAg levels in actively infected human controls.The abrupt increase was suggestive of restoration of active viralreplication.

To confirm active replication samples of serum taken from this animal at8, 10, 12 and 16 weeks were analyzed by PCR for the presence of HBV DNA.DNA isolated from 12.5 μl of mouse serum were subjected to PCR usingHBV-specific primers and amplification conditions previously described(Tipples et al. Hepatology 24:714 (1996)). All analyses were performedin blinded fashion. All four serum samples were strongly positive forthe presence of viral DNA (data not shown). This result was of specialinterest in that despite not actively replicating within its humandonor, the virus was reactivated within the immunodeficient murine host.This reactivation may have been the result of inadequate antiviralimmunity, similar to what is observed in chronic HBV carriers givenpharmacologic immunosuppression after organ transplantation (Terrault etal. Gut 40:568 (1997)).

This example thus demonstrates that human hepatocytes transplanted intochimeric, transgenic mice can support HBV viral replication.

Example 3 Establishing Primary HCV Infection

The success above in production of a chimeric animal that supports HBVreplication in the chimeric mouse supports the use of the animal as amodel of HBV. However, the vast differences between HBV and HCVdiscussed above (Background) meant that there could be no reasonableexpectation that the animal model would be susceptible to HCV infectionthrough a normal route of infection (e.g., intravenous transmission) orthat the chimeric liver could support an active HCV infection,particularly in view of the failure of others to develop HCV animalmodels and the rarity of cell systems for HCV. The comparative successwith HBV animals models and the repeated failures of others with HCVanimal models indicate that one can not simply extrapolate from HBV toHCV. Thus, an attempt was made to establish a primary HCV infection inmice with chimeric livers using virally-infected human serum.

Seven littermates were transplanted at 7 days of age with humanhepatocytes isolated from a patient serologically-negative for both HCVand HBV infection. After confirming initial graft function in 5/7animals at 6 weeks post-transplant, all mice were inoculatedintravenously with 0.25 mL of human serum obtained from an unrelatedHCV-positive donor. The HCV-positive status of the human serum donor wasconfirmed positive for HCV RNA by PCR, with viral titers of 1×10⁷ copiesper ml serum. Thus, each mouse was inoculated with approximately 2.5×10⁶viral particles. Serum samples taken from all seven mice at 11, 12 and13 weeks post-transplant (5, 6 and 7 weeks post-infection) were analyzedfor the presence of HCV RNA by RT-PCR analysis using the Cobas Amplicorsystem (Roche Diagnostics), according to the manufacturer'sinstructions. Two nontransplanted mice served as mock-infected controls.

Of the five animals with good initial engraftment, four showed thepattern of transient graft function and again one animal demonstrated HAlevels at maximal intensity over all measured timepoints. All threesamples taken from the animal with sustained human chimerism asreflected by persistent human albumin levels in serum were stronglypositive for HCV RNA, and persistently positive at weekly intervals to36 weeks. RT-PCR analysis was uniformly negative for animals negativefor the Alb-uPA transgene or that only transiently expressed the HAmarker for the transgene. As 6 animals were negative for HCV RNA, thepossibility of the positive RT-PCR signals in the seventh animaloriginating from residual virus from the inoculum is remote. Thisexample supports the conclusion that this animal had developed and at 23weeks post-transplantation and 20-weeks post-infection, is propagatingan active HCV infection at 1.2×10⁵-1.8×10⁵ virion/ml serum.

This series of experiments establishes the capacity of theSCID-beige/Alb-uPA transgenic mouse to generate and sustain a chimerichuman liver for prolonged and perhaps indefinite periods of time aftertransplantation of human hepatocytes. These chimeric organs can beinfected de novo with HCV-positive human serum, and can supportlong-term replication (e.g., for a period of weeks or months as opposedto a few days) of human-specific hepatotrophic viruses at levels thatcan be equated to clinical levels in humans. HCV viral particles can bedetected in serum, blood, or other blood-derived fraction by standardtechniques, which techniques can be automated to facilitate more rapidscreening. For example, the samples from the HCV-infection host can bediluted with known noninfected serum (e.g., about two to four folddilution), to provide a sample volume adequate for use in an automatedmachine, and provide signal strengths in the assays indistinguishablefrom random human samples.

Long-term replication of HCV in the model of the invention (e.g., for aperiod longer than about 4 weeks, generally longer than about 12 weeks,e.g., about 3 months to 6 months or more) allows for the use of themodel in the testing of drugs over extended periods of time, whichperiod may be necessary for adequate drug development. For example, theeffect of administration of interferon-α (particularly interferon-α2b),an anti-HCV therapy, is generally only detectable in humans after about12 weeks of therapy. In an animal model that sustained viral replicationfor only a few days or weeks and/or exhibited inconsistent viralproduction, it would be difficult or impossible to determine if changesin viral titers were due to a candidate therapeutic or to normalfluctuations in titer inherent in the animal model. The presentinvention provides a model that avoids this problem.

In summary, to the best of the inventors' knowledge, this is the firstreport of a non-primate animal model that is susceptible to HCVinfection by a normal route of infection. The model is clinicallyrelevant (e.g., can be infected by a normal route of infection, andsupports persistent HCV infection similar to that observed in humans),can be produced regularly and reliably in substantial numbers, and willallow investigators to directly explore strategies for inhibiting viralreplication in vivo.

Example 4 HCV Infection of Alb-uPA Mice

In this Example, the work above was expanded further to demonstrate thatthe animal model of the invention can support replication of HCV.

Methods and Materials

The following Methods and Materials were used in this example.

Development of scid/Alb-uPA strain. Animals were housed invirus/antigen-free conditions, and cared for in accordance with theguidelines established by the Canadian Council on Animal Care (1993).Approval for animal experimentation was obtained from the University ofAlberta Animal Welfare Committee.

Hemizygous Alb-uPA mice (strain TgN(Alb1Plau)144Bri, The JacksonLaboratory) were crossed with homozygous scid-bg mice (strainC.b-17/GbmsTac-scid-bgN7, Taconic Farms), and progeny carrying theAlb-uPA transgene were identified by PCR analysis of genomic DNAextracted from tail biopsies (Jackson Laboratories technical support).Through backcrossing, the scid trait was bred to homozygosity asconfirmed by quantification of total serum IgG using a sandwich ELISA.Animals with >1% of normal serum IgG were euthanized.

Isolation and purification of human hepatocytes. Ethical approval foruse of human tissue was obtained from the University of Alberta Facultyof Medicine Research Ethics Board; informed consent was obtained fromall hepatocyte donors. Segments of human liver tissue (15-20 cm³) wereobtained from regions of hepatic resection specimens which wouldnormally be discarded after pathologic examination; the majority ofoperations were performed for intrahepatic malignancies.

After rapid cooling of resected specimens, hepatocytes were isolated andpurified by standard two-step collagenase-based perfusion (SeglenMethods Cell Biol. 13:29-83 (1976); Ryan et al. Surgery 113:48-54(1993)) using 0.38 mg/mL Liberase CI (Boehringer-Mannheim) in thecollagenase perfusate. After purification, cells were washed andsuspended in Belzer-UW solution (DuPont) at 0° C. for short-term storageprior to transplantation. Cell counts and viability were confirmed byhemocytometer and trypan blue exclusion; final viability was routinely>80%.

Transplantation of human hepatocytes. Recipients (5-14 days old) wereanesthetized with halothane/O₂, and a small left flank incision wasmade. Under operating magnification, 1×10⁶ viable hepatocytes wereinjected into the inferior splenic pole with a 27 g butterfly injectionset (Abbott), with a single sterile titanium clip placed across theinjection site for hemostasis. The spleen was replaced and the flankincision closed in two layers.

Detection of HA in mouse serum by immunoprecipitation and Western blot.Mouse serum (20 μl) was incubated with monoclonal anti-HA antibody(Clone HSA-9, Sigma) and antigen-antibody complexes collected withprotein G-agarose beads (Boehringer-Mannheim). Under reducingconditions, immunoprecipitates were separated by SDS-PAGE andtransferred to nitrocellulose. Western blots were prepared using abiotinylated monoclonal anti-HA antibody (Clone HSA-11, Sigma), with astreptavidin-HRP conjugate and chemiluminescent substrate (Pierce) forsignal detection.

Determination of zygosity of the Alb-uPA transgene. Mouse DNA (3 ug) wasdigested with PvuII, size fractionated on 0.7% agarose gel, transferredto Hybond-N+membrane (Amersham Life Science), and hybridized to a[³²P]-labeled probe from the final intron of the uPA gene (positions7312-7920, GenBank accession M17922). A band of 2.88 kb was derived fromuPA transgenes (T) and a 2.53 kb band from endogenous uPA genes (E);hybridization was quantified with a Fuji phosphoimager and Image GaugeSoftware.

Immunohistochemistry. Mouse liver biopsies were fixed in 10% formalinand embedded in paraffin. Sections 5μ thick were stained withhematoxylin and eosin (H&E) in standard fashion. Selected sections weretreated with an endogenous avidin/biotin blocking kit (ZymedLaboratories, INC.) and immunostained with a monoclonal anti-humanhepatocyte antibody (DAKO, 1:20 dilution); bound antibody was detectedusing the Super Sensitive Immunodetection System (BioGenex).

Protein dot-blot assay for quantitation of HA production. Samples ofmouse serum (2 μl) were incubated for 5 min at 100° C. in 40 μl reducingbuffer, and 2 μl aliquots were blotted in triplicate ontonitrocellulose. Dried membranes were soaked in transfer buffer, blockedwith 3% PBS-Tween, and prepared as Western blots. Chemiluminescence wasquantified using a STORM phosphoimager, from a standard curve preparedon each blot.

Quantitative analysis of positive strand HCV RNA in mouse serum.Quantitative HCV analysis was performed in blinded fashion by theAlberta Provincial Laboratory of Public Health (Edmonton, Alberta,Canada), or the Canadian Center for Disease Control (Winnipeg, Manitoba,Canada). Analysis was performed on serum samples using the CobasAmplicor HCV Monitor system (Roche Diagnostics) according tomanufacturers instructions.

Detection of negative-stranded HCV RNA by thermostable rTth reversetranscriptase RNA PCR. Total RNA was isolated from mouse liver biopsiesor infected human serum using TRIZOL (Gibco BRL). RT-PCR was performedusing a thermostable rTth reverse transcriptase RNA PCR kit (PerkinElmer) according to manufacturer's instructions. Positive-strand RNA wasdetected with an antisense (5′-CTCGCAAGCCCCTATCAGG-3′ (SEQ ID NO:1))primer and negative-strand with a sense (5′-GAAAGCGTCTAGCCATGGCGT-3′(SEQ ID NO:2)) primer for reverse transcription 14. Strand-specific cDNAwas amplified by adding the other primer to target a 240-base pair (bp)region of the 5′ non-coding region (NCR) and subjected to 35 cycles at95° C. for 30 s, 66° C. for 45 s and 70° C. for 90 s, followed by 70° C.for 5 minutes. Reaction products were loaded onto a 2% agarose gel,transferred to Hybond-N+ nylon membrane (Amersham Pharmacia Biotech) andhybridized with an α-³²P-labelled DNA probe for HCV 5′ NCR at 42° C.overnight.

Detection of negative-stranded HCV RNA by RNase protection assay. TotalRNA was isolated from mouse liver using Trizol Reagent (GIBCO/BRL) andfrom HCV-infected human serum using QIAamp Viral RNA Mini Kit (Qiagen),each according to manufacturer's protocol. Extracted RNA was probed with32P-labeled, gel-purified antisense riboprobe (detection of (+) strand),sense riboprobe (detection of (−) strand), and/or β-actin antisenseriboprobe.

Plasmid Constructs. Three plasmid constructs were prepared for in vitrotranscription of truncated HCV RNA. HCVPfix/KS+ is a constructoriginally developed in our lab for expression studies of HCV serineproteinase. Total RNA prepared from fresh serum obtained fromHCV-infected patients (Chomczynski et al. Anal Biochem 162, 156-159(1987)) was denatured at 95° C. for 5 min. cDNA synthesis was performedin a 20 μl reaction volume with AMV super reverse transcriptase(Molecular Genetic Resources) at 42° C. for 90 min. The antisenseoligonucleotide primer used was 5′-TCTCTGTCGACTCACTGGGGCACTGCTGGTGG-3′(SEQ ID NO:3) (3′primer). PCR was performed in a total volume of 100 μland contained 2 μl of the final cDNA reaction mixture, 20 mM Tris-HCl(pH 8.4), 50 mM KCl, 1.5 mM MgCl2, 200 μM of each deoxyribonucleosidetriphosphate, 0.5 μM of 3′primer and 5′primer(5′-GGAATTCGCGACGACGATGACAAGGCACCCATTACGGCGTATGCCCAGCAGACAAGGGGCCTCTT-3′ (SEQ ID NO:4)), and 2.5 U Taq DNA polymerase(GIBCO/BRL). The 5′ primer added an enterokinase cleavage site, and theentire 623 bp fragment was cloned into the Eco R1 and Sal 1 sites ofpBluescript KS+ (Stratagene, PDI).

The remaining two plasmids were constructed using a PCR strategy toobtain a region of the highly conserved 5′ noncoding region (NCR) of HCVRNA from pCV-H77C (Masayuki et al. J. Proc Nat Acad Sci USA 94,8738-8743 (1994)). PCR components were as above except the template usedwas 100 ng pCV-H77C and primers were 5′-GAAAGCGTCTAGCCATGGCGTTAG-3′ (SEQID NO:5) (5′ primer) and 5′-GGCACTCGCAAGCACCCTATCAGGC-3′ (SEQ ID NO:6)(3′ primer). Both orientations of the 245 bp product were TA-cloned intopCR2.1 TOPO using a commercially available TOPO TA Cloning kit(Invitrogen) to generate pCR 2.1/NCRsense and pCR 2.1/NCRantisenseplasmids. All clones were confirmed by DNA sequencing (University ofAlberta DNA Core Facility).

Preparation of Riboprobes and Truncated HCV RNA Transcripts. Riboprobeswere prepared by in vitro transcription using T7 RNA polymerase(Promega) according to the manufacturer's instructions, in the presenceof [32P]UTP. For detection of (+) strand HCV RNA, a 383-nt antisenseriboprobe was transcribed from Kpn I digested pCR 2.1/NCRantisense andfor (−) strand, a 636-nt sense riboprobe was transcribed from Sal1digested HCVPfix/KS+. For detection of β-actin, linearizedpTRI-Actin-Mouse (AMBION) was used to generate a 304 nt riboprobe. Todemonstrate specificity of strand-specific detection of HCV RNA,radioinert sense and antisense riboprobes were also prepared. Briefly,pCR2.1/NCR sense/antisense were digested with Kpn I and HCVPfix/KS+wasdigested with Sal I prior to in vitro transcription using T7 RNApolymerase. Additionally, HCVPfix/KS+was linearized with Eco R1 and invitro transcribed using T3 RNA polymerase (Promega). All in vitrotranscription reactions were 1 hour at 37° C. followed by treatment withRNase-free DNaseI (RQ1 DNase, Promega) for 30 min at 37° C. Labeledriboprobes were gel purified and radioinert HCV RNA riboprobes wereprecipitated with 2.5 volumes of absolute ethanol after adding 0.5volumes of 7.5 M ammonium acetate. Integrity of radioinert in vitrotranscribed RNAs was evaluated by electrophoresis through a 1% agarosegel. RNA samples were denatured and hybridized overnight at 42° C., andRNase digestion was performed using an RNase protection assay kit(AMBION RPA III Kit). Products were resolved on a 5% polyacrylamide gelcontaining 8 M urea and exposed to Kodak X-Omat AR film.

Production of Transgenic Mice and Transplantation of Human Hepatocytes

Mice carrying Alb-uPA were crossed with animals from a C.b-17/scid-bglineage, and through selective backcrosses bred the scid trait tohomozygosity. In initial experiments, homozygous scid animals carryingthe Alb-uPA transgene in hemizygous fashion were crossed, and litters of4-12 day-old progeny were transplanted intrasplenically with 0.5-1×10⁶freshly isolated viable human hepatocytes. Human albumin, producedexclusively by human hepatocytes, was employed as an indicator of graftfunction.

In a pilot study of 36 transplants, a strong HA signal at 4-5 weekspost-transplant was demonstrated in the serum of 19 recipients. HA bandswere detected as early as two weeks post-transplant and increased inintensity over the 4-6 week timepoints suggesting graft expansion (FIG.5). Blinded genotype analysis revealed that all strongly HA-positiveanimals carried Alb-uPA whereas the remainder did not.

Despite initially strong HA signals, some graft recipients hadextinction of signal at around 14 weeks, while a second subsetmaintained strong signals beyond 30 weeks; representative results areshown in FIG. 7. As these graft recipients were progeny of heterozygouscrosses, the divergence in graft survival as the result of zygosity ofthe Alb-uPA transgene was tested. Using a [³²P]-labeled probe derivedfrom the final intron of the uPA gene, transgenic and endogenous uPAwere distinguishable by Southern blot analysis, and the signal ratiocould be used to determine the zygosity of the transgene array (FIG. 6).Genomic DNA analysis confirmed that animals demonstrating sustainedhuman engraftment were homozygous for Alb-uPA, whereas the subset withfailing graft function were hemizygous.

Examination of sections from transplanted homozygote livers revealedlarge nodules of hepatocytes arranged in typical cord-like structures.Within nodules, hepatocyte cytoplasm and nuclei appeared histologicallynormal in contrast to surrounding tissues, where cells were obviouslysmaller, with vacuolated cytoplasm and pyknotic nuclei. To delineatehuman cells, we immunostained sections with a monoclonal anti-humanhepatocyte antibody which intensely stained control human liver but hadno substantial cross-reaction with non-transplanted homozygous mouseliver. This demonstrated that the nodules were clearly of human origin,expanding outward into and compressing surrounding murine-derivedtissues. While the large human nodules contained healthy hepatocytes,all biliary tract and portal structures appeared to be host-derived.

HCV Infection

With evidence of prolonged human engraftment, mice were infected withserum from HCV-infected human donors. Non-infected human hepatocyteswere transplanted into 27 offspring from heterozygous crosses, and at 6weeks after transplantation all mice were inoculatedintravenously±intraperitoneally with 0.25 ml of human serum obtainedfrom one of two unrelated HCV-positive donors (viral genotypes 1a and6a). Selected serum samples between 3 and 40 weeks after inoculationwere analyzed for the presence of positive-stranded HCV RNA by RT-PCR;results of the experiment are summarized in Table 2. Graft duration wasdefined as the period of HA detectability by immunoprecipitation/Westernblot procedure.

TABLE 2 Infection of homozygous mice with human HCV Median Graft Alb-uPAInitial HA Duration Genotype n Signal (weeks) HCV RNA (RT-PCR) −/− 8None* 0 0/8  +/− 15 Strong 15.5 0/15 +/+ 4 Strong 30.5† 4/4‡ *3/8animals had a single weak HA signal at 5 week timepoint only †p < 0.001vs. hemizygotes and wild-type by Kruskal-Wallis test ‡p < 0.001 vshemizygotes and wild-type by Pearson Chi-square test

All 8 wild-type controls had no evidence of initial graft function andwere persistently negative for HCV RNA. Hemizygous animals had initiallystrong HA signals, but progressively lost signal intensity over time toa median graft duration of 15.5 weeks; (+) stranded HCV RNA was notdetected in any of these animals over multiple timepoints. In sharpcontrast, all four animals homozygous for the Alb-uPA transgenedemonstrated sustained human chimerism (median 30.5 weeks) and werepositive for HCV RNA by serum RT-PCR analysis. Quantitative HCV RNAanalysis revealed viral levels ranging from 1.4×10³ to 1.4×10⁶ RNAcopies/ml, well within the range of infected humans. Successfulinfections were established with both genotypes of viral inoculum andduration of infection ranged from 10-21 weeks in this initial cohort offour animals.

HCV Infection of Animals Homozygous or Hemizygous for the Alb-uPATransgene

While positive-stranded HCV RNA was persistently demonstrable inhomozygous animals, HCV RNA was undetectable in hemizygotes. Wehypothesized that hemizygotes fail to support HCV replication atdetectable levels as a result of diminished initial engraftment andearlier graft loss. To test this hypothesis, a protein dot-blot assaywas developed using chemiluminescence and phosphorimaging to moreaccurately quantify HA production. After transplanting 1×10⁶cryopreserved human hepatocytes from a single human donor into 21recipients (15 homozygotes and 6 hemizygotes), randomly selected animalswere sampled for quantitative HA analysis and/or sacrificed forimmunohistochemical analysis. Results of this experiment are shown inFIG. 8.

While hemizygous and homozygous animals initially had similar HA signalintensities, by 5-6 weeks a clear dichotomy became apparent and by 10-12weeks HA signals in homozygous mice were more than an order of magnitudehigher than hemizygotes (FIG. 8). Random liver sections from homozygousand hemizygous recipients sacrificed at selected points aftertransplantation were immunostained with a monoclonal anti-humanhepatocyte antibody to estimate the percent replacement of murine liverwith human tissue. These immunohistochemical data confirmed the proteindot-blot findings, with human cells occupying substantial portions(>50%) of cross-sectional liver area in homozygous animals. In distinctcontrast, examination of multiple sections of tissue from heterozygousrecipients revealed only minimal evidence of human engraftment.Together, these studies suggest a substantial advantage in both themagnitude and duration of human hepatocyte engraftment for homozygousAlb-uPA recipients compared to their heterozygous counterparts.

By transplanting into the progeny of heterozygous crosses, successfulinfections were established in 4/27 mice, all homozygotes. This successrate would make the model too cumbersome for routine use. As a result ofthe quantitative advantage in graft size ascribed to homozygous mice,the breeding colony was shifted towards exclusive production ofhomozygous Alb-uPA mice. Using the dot-blot assay described above toscreen early for high-level hepatocyte engraftment (HA levels >250μg/ml), ˜75% of HCV-inoculated animals developed persistent viral titers>3×10⁴ copies/ml, with many >10⁶ copies/ml. The remainder of the viralstudies were performed in homozygous recipients.

Confirmation of Long-Term Persistence of HCV

Long-term persistence of viral titers in humans is the result of ongoingactive proliferation. In immunocompromised chimeric animals, however,one might ascribe HCV persistence to slower viral elimination ratherthan true infection and replication. Five homozygous graft recipientswere inoculated with 250 μl of infected human serum (genotype 3a;2.95×10⁶ viral RNA copies/ml); each animal received therefore aninoculum of 7.38×10⁵ RNA copies. Results of this experiment are shown inFIG. 9. In 3/5 recipients, viral titers increased by 16-, 27- and36-fold over the initial inoculum by 5 weeks after inoculation; in theremaining 2 recipients titers increased modestly over 5 weeks (1.6- and4.3-fold).

Ongoing detection of positive-stranded HCV RNA has been confirmed tobeyond 15 weeks after inoculation in four animals (one death after bloodsampling). The initial rise in titers coupled with persistently highviral levels at 15 weeks is consistent with viral replication ratherthan carryover artifact. In further study, a sixth chimeric mouse wasinfected with a much smaller viral inoculum (1.35×10³ RNA copies). Thetotal serum viral load at 10 weeks after infection was measured at1.33×10⁶ copies, a 1000-fold increase. A nonproductive “interaction”could not reasonably sustain a 3-log increase in viral load, stronglysupporting the occurrence of viral replication.

HCV is a positive-stranded RNA virus replicating through anegative-stranded intermediate; detection of (−) strand HCV RNA withinthe liver constitutes proof of replication. To reduce the risk of falsepositive results (Lanford et al. Virology 202:606-614 (1994)), (−)strand analysis was performed using two separate but complementarytechniques.

Eight homozygous graft recipients inoculated with 5×10⁵ copies of viralRNA from freshly obtained human serum were confirmed to have (+) strandHCV RNA at 3-4 weeks post inoculation. Samples of liver tissue wereobtained by 50% partial hepatectomy at 2-5 weeks post-inoculation in sixanimals and 12-13 weeks in the remaining two.

Analysis for (−) strand HCV RNA was performed in blinded fashion by anindependent laboratory (A.R.) using a thermostable rTth reversetranscriptase RNA PCR protocol and strand-specific primers. The results,shown in FIGS. 10A-10C, confirmed the production of the HCV replicativeintermediate (negative-stranded viral RNA) within the livers oftransplanted and infected homozygous Alb-uPA mice. Letter designations(A through J, specified below) in FIGS. 10A-10C are control samples;number designations (1 through 10) represent individual RNA samplesisolated from the livers of ten homozygous mice which were transplantedand then inoculated with HCV-infected human serum.

FIG. 10 A shows detection of (+) strand RNA (upper panel) or (−) strandRNA (lower panel) by thermostable rTth reverse transcriptase RNA PCRprotocol with strand-specific primers. A is a wild-type control mouse,nontransplanted, noninfected; B is a heterozygous transplanted mouseinoculated with HCV; C is a homozygous transplanted mouse, notinoculated with HCV; D is serum taken from an infected human; E is astandard DNA ladder; F represents binding of labeled probe to target DNAsequences generated from (+) strand (upper panel) or (−) strand (lowerpanel) viral RNA; G is mouse liver RNA (10 μg) doped with serum RNA froman HCV-positive human; H is mouse liver RNA (10 μg) doped with 10⁶copies radioinert antisense (upper) or sense (lower) riboprobe; I ismouse liver RNA (10 μg) doped with 10⁶ copies radioinert sense (upperpanel) or antisense (lower panel) riboprobe; J is riboprobes hybridizedwith 10 μg mouse liver RNA, all subsequent steps identical exceptaddition of RNase. Fragments in this lane represent undigested riboprobe(arrow), with expected lengths greater than those of correspondingfragments protected by hybridization to their targets. Replication ofthe HCV genome is clearly seen in 5/9 animals assayed by this method.

FIG. 10B shows the results of a dilution series analysis of selectedanimals using the thermostable rTth reverse transcriptase RNA PCRprotocol. Both (+) and (−) stranded RNA are detectable over 2-3 logdilutions. In this experiment only, (−) stranded viral RNA was notdetected in mouse 5 although it was seen earlier and was confirmed laterin multiple RPA analyses. The results of detection of (+) strand HCV RNA(upper panel), (−) strand HCV RNA (middle panel) or β-actin RNA (lowerpanel) by RNase protection assay are shown in FIG. 10C. Control lanesare as designated above; mouse 10 was analyzed only by the RPA method.This assay correlated with the above data 5/6 animals, confirmingpresence of the (−) strand in 3/4. Failure to detect (−) stranded RNA inmouse 6 is likely due to the reduced sensitivity of the RPA assay.Immunohistochemical analysis.

To confirm localization of HCV within transplanted mouse livers,sections of liver taken from homozygous mice which had been transplantedwith human hepatocytes and then inoculated with HCV-infected human serumwere immunostained with a monoclonal antibody against the NS3-NS4 regionof the viral polyprotein. Control sections of human liver show agranular cytoplasmic appearance, with exclusion of staining from thenuclei (FIG. 11A). Areas of fibrosis and portal triad structures did notstain positive for NS3-NS4. Although the majority of hepatocytes didstain positively, there were areas of sparing. Control sections fromnontransplanted mouse livers did not show any evidence at all ofstaining (not shown). Experimental sections taken from transplanted andinfected mice (FIG. 11B) showed areas of hepatocyte staining which weresimilar in cytoplasmic granular appearance to control human sections,although at a slightly reduced staining intensity. Thisimmunohistochemical finding provides evidence that HCV does truly infecthuman hepatocytes within the chimeric liver of a transplanted Alb-uPAmouse.

Conclusion.

These separate and independently-performed assays clearly demonstratepresence of negative-stranded HCV RNA within chimeric livers sampled at2-5 weeks post-inoculation. Experiments with sequential weekly analysisby quantitative RT-PCR (FIG. 9) demonstrated a rapid rise in HCV serumtiter at weeks 2-4 after inoculation, corresponding to maximal rates ofviral replication within the liver; this would be expected to beparalleled by maximal amounts of (−) stranded viral RNA. This mayexplain why the (−) strand is detectable earlier in infections (5/6animals sampled at 2-5 weeks) rather than later (0/2 sampled at 12-13weeks). Taken in combination, these data conclusively support activeviral replication in this animal model. Furthermore, HCV infection ischronic. Most recently, the inventors have demonstrated an animal modelof the invention based upon a chimeric, Alb/uPA transgenic mouse havinga functional human hepatocyte graft (as determined by detection of highalbumin serum levels at 35 weeks post-transplant (greater than about 800units by dot blot)) and high titer HCV in serum at 35 weekspost-transplant (1.7×10⁵ copies/ml).

Serial Passage of HCV Infection

After confirming replication, serial passage of HCV infection from mouseto mouse was attempted. Fresh serum from a human donor (250 μl; 4.75×10⁵viral RNA copies) was inoculated intraperitoneally into a naïve chimericmouse; at four weeks after inoculation, viral titers were 1.76×10⁶copies/ml. Serum taken from this mouse (125 μl; ˜2.19×10⁵ RNA copies)was inoculated intraperitoneally into a second naïve chimeric mouse,which developed titers of 1.75×10⁴ copies/ml at four weeks afterinoculation. Serum from this first-passage recipient was then inoculated(100 μl; ˜1.75×10³ RNA copies) into a third naïve chimeric mouse. Atfive weeks after inoculation, this second-passage recipient had viraltiters of 3.42×10⁶ copies/ml. If one assumes the null hypothesis thatreplication does not occur but rather the initial human inoculumpersists, this second-passage recipient would have received ˜6000 copiesof virus from the initial inoculum (4.75×10⁵ viral copies×1:8dilution×1:10 dilution, assuming mouse serum volume ˜1000 μl); thesecond-passage recipient had 576× more measured viral RNA than wouldhave been received from the original human inoculum. Serum from thissecond-passage recipient (30 μl) was inoculated into two additionalnaïve mice, both of whom subsequently developed HCV infections(third-passage recipients; quantitation pending). Serial transmissionhas thus far been demonstrated in 7 animals including 2 animals afterthree generations of passage. This transmission fromhuman→mouse→mouse→mouse→mouse represents both replication of the HCVgenome and production of fully-infectious particles.

These experiments establish that homozygous scid/Alb-uPA mice withchimeric human livers can be infected de novo with HCV-positive humanserum, support HCV replication at clinically relevant titers, and arecapable of transmitting this infection to other chimeric mice.Successful infections have been established with viral genotypes 1a, 1b,3a and 6a, with rapid increases in viral RNA titers to levels easilydetectable by standard commercial assays. Homozygosity of Alb-uPA iscritical to successful establishment of viral infection, and by usinghomozygotes as recipients, coupled with early screening of graftfunction by dot blot analysis, HCV infections are routinely establishedin ˜75% of all inoculated animals.

The transplantation procedure requires basic microsurgical equipment andtechnical skills. In our hands a transplant, including anaestheticinduction and recovery time, takes 5-6 minutes per animal. While accessto human hepatocytes may be limiting for some investigators, the yieldsfrom hepatocyte isolations in our laboratory average 2-3×10⁸ viablehuman cells. The ability to cryopreserve surplus cells allows forefficient utilization, as well as transportation to centers withouthuman tissue access. While success rates have been lower aftertransplanting cryopreserved hepatocytes, prescreening these recipientswith dot-blot hybridization has allowed for their efficient use in HCVstudies, with success in viral infection in approximately 50% of animalswith dot blot>250 units at time of inoculation.

Example 5 Human Umbilical Cord Blood Cells as Source of Cells forTransplantation

In the current literature, human stem cells are proven to bepluripotent. They have the ability to regenerate into hematopoieticcells as well as hepatocytes given the proper combination of conditionsand stimuli. Human stem cells have been shown to have the ability torepopulate the hematopoietic cells in NOD/SCID mice (see, e.g., Bhatiaet al. J Exp Med 186(4):619-24 (1997); Bhatia et al Proc Natl Acad SciUSA 94(10):5320-5 (1997); Larochelle et al. Nat Med 2(12):1329-37(1996). With regards to liver repopulation, however, clinical studieshave used stem cell transplants in pediatric hepatoblastomas toregenerate liver.

Human cord blood is a rich source of stem cells. In addition, they havebeen reliably cryopreserved and cell integrity is preserved post-thaw.Human cord blood is also much more readily available. Because of thelimited availability of fresh liver tissue, an alternate source of humanhepatocytes is useful in the development of the animal model of theinvention. The human cord blood cells transplanted into theSCID.bg/Alb-uPa mice of the invention can regenerate into viable humanhepatocytes, and engraft and develop a chimeric mouse/human liver. Inaddition, the cells can repopulate the immune system of ourSCID.bg/Alb-uPa mice.

Materials and Methods

Following the protocol described herein, SCID.bg/Alb-uPa mice aretransplanted with human hepatocytes at 10-14 days of age. Instead ofhuman hepatocytes, 5 million human cord blood monocytes (source of stemcells) are transplanted via intrasplenic injection. The protocol oftesting mouse sera for the presence of human albumin via dot blot atfour weeks post-transplant is followed as described herein.

In addition, to determine whether the immune system has also beenrepopulated, a peripheral blood smear is performed at 4 weeks of age tolook for the presence of lymphocytes. At eight weeks, the serum istested for the presence of human IgG via ELISA and the presence of CD4+and CD8+ cells with FACS analysis. To test functionality, PHAstimulation is performed.

Example 6 Interferon Alpha-2b Treatment In Scid/uPA Mice Infected WithHCV

The HCV animal models of the invention can be used to screen foranti-HCV activity of candidate chemotherapeutics. Interferon alpha-2b isknown to have anti-HCV activity. Thus, treatment of HCV-infected mice ofthe invention with recombinant interferon alpha-2b will result in asignificant decrease in the levels of HCV RNA.

Methods and Materials:

Animals: Human hepatocytes were isolated from pieces of human livertissue obtained from the operating theater using continuous perfusionwith collagenase (Liberase HI, Boehringer Mannheim). Homozygote SCID/uPAmice were transplanted with 0.5×10⁶ to 1.0×10⁶ fresh human hepatocytesvia intrasplenic injection at 10-15 days of age. At 4 weekspost-transplant blood was drawn and assayed for human albumin (HA)concentration using a quantitative dot blot assay. Micedemonstrating >200 ug/ml of HA were considered to have a successfulgraft and used for this experiment.

HCV infection: At 8 weeks post-transplant, mice were injectedintraperitoneally with 50 μl of serum from a HCV positive livertransplant patient, genotype 3. The serum was stored at −70 degreesCelsius and thawed out at time of inoculation. The patient serumdemonstrated HCV RNA levels of 2.56×10⁵ IU/ml. All HCV quantitation wasperformed by the Provincial Laboratory at the University of AlbertaHospital using the Cobas Amplicor HCV Monitor version 2.0 (RocheDiagnostics).

Interferon administration: The mice were divided into 3 treatmentgroups: group 1=controls (n=5), group 2=interferon-α2b (IFN) 135 IU/g/d(n=1), group 3=1350 IU/g/d (n=2). Treatment was started 2 weeks afterHCV inoculation. The IFN (or an equivalent volume of normal saline) wasinjected IM for 15 consecutive days. Blood was drawn to assay for HCVRNA levels and graft function at the start of treatment, at the end oftreatment, and 2 and 4 weeks after treatment had stopped.

Animals received human hepatocyte transplants and, after confirmation ofsatisfactory engraftment by serum dot-blot assay for human albuminof >250 units, were injected with 100 μl IP of serum from a humancarrier of genotype 3 HCV. Baseline values are viral copies/ml mouseserum from 2 weeks post HCV injection, when prior studies revealed thegreatest absolute rise in HCV copies. Interferon therapy was begun atbaseline in 3 animals at dosages of 135 (n=1), or 1350 (n=2) IU/grambody weight/day for 2 weeks of treatment. Week 2 is HCV titre by RT-PCRat end of interferon therapy, while week 4 is 2 weeks after therapy;assay was by the Roche Amplicor kit run by the Provincial Laboratory ofPublic Health of Alberta. Samples were blinded and interspersed withhuman serum samples from clinical analyses. Assay sensitivity is 6001U/ml or approximately 1.2×10³ viral copies/ml. The results are shown inthe Table 3 below (“E” indicates the exponent value).

TABLE 3 Affect of IFN upon HCV Infection in the Animal Model of theInvention HCV titre (RT-PCR) Treatment Baseline week 2 week 4 Control2.7 × 10E3 2.5 × 10E4 6.6 × 10E3 Control 2.4 × 10E4 7.5 × 10E5 1.4 ×10E6 Control 1.6 × 10E5 1.7 × 10E5  .9 × 10E5 Control 7.5 × 10E5 2.1 ×10E6 1.5 × 10E6 Control 1.2 × 10E6  >2 × 10E6 1.7 × 10E6  135 IU/g/d 2.2× 10E5 3.6 × 10E4 4.5 × 10E4 1350 IU/g/d 1.8 × 10E3 ND ND 1350 IU/g/d3.3 × 10E4 ND ND

Four of five control untreated mice demonstrate rising titres of HCVover this time period, while 1 shows stable levels. All 3 treatedanimals demonstrated decreasing viral titres with the 2 at higher dosedemonstrating viral clearance (ND=not detected).

Example 7 Passive Immunity to Hepatitis B Infection with administrationof HBIg

The HCV model described herein is based on the presence of a chimericmouse/human liver in an immunocompromised animal, as a specific example,the SCID.bg/Alb-uPa mouse. This model not only supports a replicatinghepatitis C virus, but has also supports a hepatitis B infection aswell. Because there are no currently no proven vaccinations forhepatitis C, HBV-infected mice are used to test the validity of theanimal model in testing vaccinations. There are currently both passiveand active immunizations available for HBV.

Hepatitis B Immunoglobulin (HBIg) is a developed passive vaccine to thehepatitis B surface antigen. It is developed by collecting and poolingthe plasma from positive anti-HBs donors. The final result is a hightitre anti-HBs preparation. In the clinical setting, it has limitedapplicability because of 1) partially effective 2) short half-life and3) interferes with long lasting immunity. However, in certainsituations, it has proven to be useful.

In liver transplantation, HBIg immunoprophylaxis is widely used andaccepted. It has shown to significantly reduce the recurrence rate ofHBV post-transplant in hepatitis B positive patients. Consequently, ithas reduced the morbidity in both the graft and the patient. Patientsare treated with a large bolus dose during the anhepatic stage of thetransplant. Treatment is continued for one year and the dosage isdetermined by the anti-HBs antibody titre. Post needle-stick exposure,the administration of HBIg has prevented the transmission of HBV in 80%of cases.

The animal model of the invention can be used in vaccine development. Asa control to demonstrate the usefulness of the animal model in vaccinedevelopment, a proven immunoprophylactic vaccine available for HBV isused. Through injections of hepatitis B immunoglobulin (HBIg), theSCID.bg/Alb-uPa mice will obtain passive immunity to a subsequentinoculation of hepatitis B, thus preventing and active viral infection.

As emphasized above, HBV and HCV are not comparable viruses. However,since there is currently no passive immunotherapy available for HCV, theuse of HBV and the HBIg provides an initial screen to show that animmunotherapeutic known to be effective against HBV infection iseffective in the animal model of the invention provides further evidencethat the animal model in fact provides for a valuable screening tool forpassive immunotherapy.

Materials and Methods

Following the protocol described in the Examples above, SCID.bg/ALB-uPamice are transplanted with human hepatocytes at 10-14 days of age. Fourweeks post-transplant, the mice are tested by dot blot for humanalbumin. Those animals with a strong signal are then chosen forexperimental use.

Day 0: At eight weeks of age, the mice allotted into the experimentalgroup receive a high dose intramuscular injection of HBIg (1 cc/kg)where as the control group receive a injection of normal saline.

Day 1: All mice are inoculated with 100 μL of high titre HBV serum(intraperitoneal injection).

Day 1-14: Experimental mice are treated with a maintenance dose of HBIg(comparable to that in liver transplant patients) of 0.12 cc/kg once aday. Control mice are continued on normal saline injections.

To assay the effect of HBIg, serum samples are obtained for HBV titresat 2, 4, 6, 8, 10 and 12 weeks post-HBV infection. Hepatitis B surfaceantibody is assayed on day 1 prior to HBV inoculation and again at eightweeks post-infection.

Example 8 Use of Immune Reconstituted HCV Animal Model to Analyze theImmune Response in HCV Infection

The animal model of the invention provides a valuable tool to studyhuman immune responses in context of autologous liver cells infectedwith HBV or HCV. The mice carrying human liver cells can beimmune-reconstituted with autologous peripheral blood mononuclear cells(PBMCs, 2-3×10⁷ cells/mouse), to provide a model system of HBV and HCVinfection to perform the following studies. This model can then be usedin the following exemplary ways.

The experiments described below can provide insights into the criticalrole of various components of immune system, e.g., antigen presentingcells, certain cytokines and T cells, and mechanisms underlying theimmunomodulation in chronic hepatitis infection. These studies canprovide the foundation for design and investigation of novel strategiesand novel vaccine candidates for the immunotherapy of chronic hepatitisvirus infections. These studies will also establish the mouse modelsystem as preclinical model for the evaluation of futurechemotherapeutic and/or immunotherapeutic treatment of chronic hepatitisinfections.

Evaluation of the Immune Response and Modulation in Chronic HCVInfection

HCV infected mice, which are transplanted with human liver cells andreconstituted with autologous PBMCs, are used to evaluate overall immunecell competence and/or immune suppression in context of progressive HCVinfection. A time course study is performed where splenic or lymph nodeT cells are obtained from the mice and set up in in vitro culture toexamine response against mitogens, allogeneic APCs, promiscuous Thepitopes (e.g., tetanus toxoid, PADRE peptides etc.) are evaluated by Tcell proliferation assay. In the same cultures, cytokine secretion inthe culture supernatant or intracellular production is examined. Inaddition, in these cultured cells, T cell activation markers areexamined by flow cytometry. These experiments are performed in a timecourse fashion, so T cells will be recovered from mice at various times(e.g., 1, 4, 8, 12, 16 weeks post infection and PBMCs reconstitution)and examined for their response to polyclonal stimuli as stated above.HCV virus load is also evaluated at each time point, so that overallimmune response can be correlated with virus load.

Along with polyclonal stimulus, T cell responses against known conservedHCV promiscuous helper epitopes are examined in vitro and theirstimulation correlates with virus load in time course experiments.Similarly, B cells are isolated at the same time and cultured withpolyclonal B cell stimuli, e.g., LPS, α-CD40 etc. and examined forcytokine secretion as well as overall Ig production in culture uponpolyclonal stimulation. Uninfected, PBMC reconstituted mice are used ascontrols. The overall T and B cell competencies in ongoing HCVinfections can also be evaluated.

Alternatively, mice are challenged in vivo with promiscuous HCV andnon-HCV Th epitopes at various times after infection and PBMCsreconstitution followed by examination of those peptide reactive T cellsby cytokine production, activation marker expression and proliferation.Again, in these in vivo challenge experiments, overall T cell responsesare correlated with virus load, time from infection etc. T cellsobtained from the unimmunized but immune-reconstituted and HCV infectedmice are evaluated for overall CD4/CD8 ratios, MHC molecules, and otherT cell/activation molecules and compared with uninfected but immunereconstituted mice. In alternate experiments, normal human PBMCs arepolyclonally stimulated in the presence of sera from HCV infected miceand examined for any modulation of T cell responses. Following theseexperiments, phosphorylation of various TCR molecules, Ca2+ mobilizationetc., are examined to determine the biochemical basis of any observed Tcell response defects. Additionally, we will examine whether T cellsundergo apoptosis upon stimulation.

Cytokines and Immunoregulatory Molecules in Chronic Hepatitis VirusInfections.

In order to examine the role of various cytokines (type 1 vs. 2) on HCVinfection, sera is collected from mice infected with HCV andreconstituted with PBMCs and examine for 1 vs. 2 type cytokinesprevalence. These experiments are performed in a time course manner tocorrelate cytokine production with HCV virus load, and compared withcontrol non-infected but immune reconstituted mice. On the other hand,predominant 1 or 2 type cytokines, e.g., IL-2, γ-IFN, IL-4, IL-10 andIL-12 are injected or abrogated (by injecting anti-cytokine antibodies)in these mice and their effect on virus load, T cell response topromiscuous HCV and non-HCV peptides as well as polyclonal stimulusevaluated. Additionally, progression in cytokine switch, defects incytokine production or their modulation, are evaluated immediately afterinfection or after a longer time (i.e., 2-3 months in mice). In the invitro experiments, the role of addition of certain cytokines in vitro tothe T cell responses.

Antigen Presenting Cells (Dendritic Cells, DCs) in Chronic HCVInfections and Modulation of DC Function to Provide Protective ImmuneResponses.

There is some evidence to suggest that in chronic HCV infection inhumans, dendritic cell function is impaired. The dendritic cells (DCs)are the most potent stimulators of CD4+ T cells to induce efficientimmunity. Therefore, examination of DC function in context of HCVinfection is essential to understand immune response against HCVinfection.

From the HCV infected PBL reconstituted mice, monocytes are isolatedfrom spleens or blood and cultured with GM-CSF and IL-4 to generateimmature DCs. These immature DCs are matured in presence of γ-IFN,α-IFN, LPS or α-CD40 and examined for the expression of DC activationmarkers by flow cytometry, IL-12 production in the supernatant andability to stimulate allogeneic T cells & HCV promiscuous Th epitopepresentation to autologous T cells. These experiments are performed in atime course manner, and progression in change in DC function examined asa factor of time and HCV virus load.

In additional experiments, mice carrying HCV infection and reconstitutedwith human PBMCs are injected with in vitro activated mature DCs andexamined for T cell responses and virus load.

Immunization of Promiscuous Human Helper and CTL Epitopes in HCVInfection

In numerous studies reporting Th and cytotoxic T cell (CTL) responses inchronic HCV infected individuals, a number of promiscuous Th and CTLepitopes from conserved region of HCV have been identified in vitro,suggesting that Th and CTL priming occurs in the HCV infectedindividuals. However, apparently this ongoing natural T cell response initself is not sufficient to clear the virus infection and/orreplication. The animal model of the invention can be used to evaluatevarious immunization strategies (as listed below) using knownpromiscuous Th and CTL epitopes to induce strong immune responses. Themice are evaluated for generation of CTL and Th cell responses afterimmunization. In parallel, virus load is evaluated. The animal model canthus be used to determine the appropriate modulation of Th and CTLresponses to provide immunity against HCV infection.

Immunization with antigens can examined in context of particulateformulations, e.g., liposomes; modified antigen peptides, e.g.,lipidated peptides; certain adjuvants and cytokine formulations asadjuvants or adjuncts; dendritic cells loaded with antigens; DNAmediated immunizations; T cell adoptive therapy with antigen specific Tcells expanded in vitro; and the like.

Immunogenicity of Synthetic Peptides (or Modified Lipopeptides) Derivedfrom Structural and Non-Structural Proteins of HBV and HCV

An alternative hypothesis for the failure to resolve ongoing HCVinfection in HCV chronic carriers focuses on evidence that Th and CTLresponses against these epitopes are actually not able to suppress virusreplication or clear virus infected cells, and immune responses againstother epitope determinants are necessary to generate protectiveimmunity. In order to determine novel T cell epitopes on HCVpolyprotein, initial in silico studies are performed to identifyputative Th and CTL epitopes from conserved structural as well asfunctional viral proteins. These identified epitopes are then modifiedand evaluated in the reconstituted animal model of the invention fortheir ability to generate strong T cell responses, indicating that theyare immunotherapeutic vaccine candidates.

Examination of Combined Therapeutic Approaches (Antigen-Based Vaccinesand Small Molecules that Inhibit Viral Replication and/or InduceImmunomodulation.

This approach takes into consideration the immune response in chronicHCV infection, and uses the reconstituted animal model of the inventionto identify anti-viral therapeutics that take advantage of a combinationof antigen-based therapies and small molecule-based therapy, where theresmall molecule has activity in inhibition of viral replication and/or inimmunomodulation. Combinations that provide effective suppression ofvirus replication or virus clearance are identified using HCV infected,immune reconstituted animals as described above. Vaccine candidates areevaluated in combination with cytokines (such as IL-2 or liposomal IL-2)to provide efficient immunity to suppress and/or clear HCV infection.

Example 9 Use of the Model for Evaluation of Therapies forHyperlipidemia

As discussed above, Apo B100 is an art-accepted marker for risk ofartherosclerosis that results from hyperlipidemia. In order to assessthe use of the mouse model of the invention in screening for agents thathave activity against hyperlipidemia, a mouse monoclonal antibodyspecific for human Apo B100 was used to detect production of human apoB100 production by the engrafted human cells. Serum samples werecollected from a chimeric Alb/uPA transplanted animal and from a Alb/uPAnon-transplanted animal (negative control). Human serum served as apositive control. The serum was analyzed by Western blot using ananti-Apo B100 antibody according to methods well known in the art.

As shown in FIG. 12 while antibody binding to Apo B100 was not detectedin the non-transplanted control animal (lane 3), antibody binding wasdetected in both the human serum positive control (lane 1) and thechimeric Alb/uPA transplanted animal (lane 2). These data show thatAlb/uPA mouse liver with sustained human chimerism secretes human apoB100. This observation indicates that the Alb/uPA mouse model canprovide a basis for development of selective treatments that decreasethe amount of apo B 100 from the liver and, as a consequence, decreasethe risk of cardiovascular disease and stroke via atherosclerosis.

While the present invention has been described with reference to thespecific embodiments thereof, it should be understood by those skilledin the art that various changes may be made and equivalents may besubstituted without departing from the true spirit and scope of theinvention. In addition, many modifications may be made to adapt aparticular situation, material, composition of matter, process, processstep or steps, to the objective, spirit and scope of the presentinvention. All such modifications are intended to be within the scope ofthe claims appended hereto.

1-18. (canceled)
 19. A chimeric, chimeric, immunodeficient transgenicmouse mouse, wherein the genome of the chimeric, immunodeficienttransgenic mouse comprises a polynucleotide encoding a urokinase-typeplasminogen activator polypeptide, wherein the polynucleotide isoperably linked to a mouse albumin promoter such that the polypeptide isexpressed in host mouse liver cells, and wherein the mouse is homozygousfor the polynucleotide; and the chimeric, immunodeficient transgenicmouse has a chimeric liver comprising human hepatocytes engrafted intothe mouse liver, wherein the human hepatocytes constitute at least 20%of hepatocytes in the chimeric liver.
 20. The chimeric immunodeficienttransgenic mouse of claim 19, wherein human hepatocytes constitute atleast 50% of hepatocytes in the chimeric liver.
 21. The chimericimmunodeficient transgenic mouse of claim 19, wherein human hepatocytesconstitute at least 40% to 60% of hepatocytes in the chimeric liver. 22.The chimeric immunodeficient transgenic mouse of claim 19, wherein humanhepatocytes constitute at least 90% of hepatocytes in the chimericliver.
 23. The chimeric immunodeficient transgenic mouse of claim 19,wherein the human hepatocytes are functional for at least about 8 weeks.24. The chimeric immunodeficient transgenic mouse of claim 19, whereinthe human hepatocytes are functional for at least 15 weeks.
 25. Thechimeric immunodeficient transgenic mouse of claim 19, wherein thegenome comprises a scid mutation.
 26. The chimeric immunodeficienttransgenic mouse of claim 25, wherein the genome comprises a beigemutation.
 27. A method for evaluating liver toxicity of an agent, themethod comprising the steps of: administering a candidate agent to,wherein the genome of the chimeric, immunodeficient transgenic mousecomprises a polynucleotide encoding a urokinase-type plasminogenactivator polypeptide, wherein the polynucleotide is operably linked toa mouse albumin promoter such that the polypeptide is expressed in hostmouse liver cells, and wherein the mouse is homozygous for thepolynucleotide; and the chimeric, immunodeficient transgenic mouse has achimeric liver comprising human hepatocytes engrafted into the mouseliver, wherein the human hepatocytes constitute at least 20% ofhepatocytes in the chimeric liver; and analyzing the effect of thecandidate agent upon human liver function or human liver histology;wherein a decrease in liver function or adverse alteration in liverhistopathology in the presence of the agent relative to the absence ofthe agent, indicates the agent is toxic to human liver cells.
 28. Amethod for screening candidate agents for activity in decreasing bloodlipids, the method comprising the steps of: administering a candidateagent to a chimeric, immunodeficient transgenic mouse, wherein thegenome of the chimeric, immunodeficient transgenic mouse comprises apolynucleotide encoding a urokinase-type plasminogen activatorpolypeptide, wherein the polynucleotide is operably linked to a mousealbumin promoter such that the polypeptide is expressed in host mouseliver cells, and wherein the mouse is homozygous for the polynucleotide;and the chimeric, immunodeficient transgenic mouse has a chimeric livercomprising human hepatocytes engrafted into the mouse liver, wherein thehuman hepatocytes constitute at least 20% of hepatocytes in the chimericliver; and analyzing the effect of the candidate agent upon serum humanapoB100 lipoprotein; wherein a detection of a level of human apoB100following candidate agent administration that is decreased relative to alevel of human apoB100 prior to candidate agent administration indicatesthe candidate agent has activity in decreasing blood lipids.
 29. Themethod of claim 28, wherein human hepatocytes constitute at least 50% ofhepatocytes in the chimeric liver.
 30. The method of claim 28, whereinhuman hepatocytes constitute at least 40% to 60% of hepatocytes in thechimeric liver.
 31. The method of claim 28, wherein human hepatocytesconstitute at least 90% of hepatocytes in the chimeric liver.
 32. Themethod of claim 28, wherein the human hepatocytes are functional for atleast 8 weeks following transplantation into the mouse.
 33. The methodof claim 28, wherein the human hepatocytes are functional for at least15 weeks following transplantation into the mouse.
 34. The method ofclaim 28, wherein the genome comprises a scid mutation.
 35. The methodof claim 28, wherein said analyzing comprises analyzing the effect ofthe candidate agent upon human hepatocyte function.
 36. The method ofclaim 28, wherein said analyzing comprises analyzing the effect of thecandidate agent upon human hepatocyte histology.
 37. The method of claim28, wherein said human hepatocyte function is analyzed by assessinglevels of human alpha-1 antitrypsin.